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Sample records for somatic embryogenesis induction

  1. Somatic Embryogenesis Induction and Plant Regeneration in Strawberry Tree (Arbutus unedo L.).

    PubMed

    Martins, João F; Correia, Sandra I; Canhoto, Jorge M

    2016-01-01

    Somatic embryogenesis is a powerful tool both for cloning and studies of genetic transformation and embryo development. Most protocols for somatic embryogenesis induction start from zygotic embryos or embryonic-derived tissues which do not allow the propagation of elite trees. In the present study, a reliable protocol for somatic embryogenesis induction from adult trees of strawberry tree is described. Leaves from in vitro proliferating shoots were used to induce somatic embryo formation on a medium containing an auxin and a cytokinin. Somatic embryos germinated in a plant growth regulator-free medium. PMID:26619869

  2. Dynamics of the concentration of IAA and some of its conjugates during the induction of somatic embryogenesis in Coffea canephora

    PubMed Central

    Ayil-Gutiérrez, Benajmín; Galaz-Ávalos, Rosa María; Peña-Cabrera, Eduardo; Loyola-Vargas, Victor Manuel

    2013-01-01

    Most of the somatic embryogenesis (SE) process requires the presence, either before or during the embryogenic process, of at least one exogenous auxin. This exogenous auxin induces the presence of endogenous auxins, which appears to be essential for SE induction. We found that during the preincubation period of SE in Coffea canephora, there is an important increase in both free and conjugated indole-3-acetic acid (IAA), as well as indole-3-butyric acid. This increase is accompanied by an increase in the expression of YUCCA (CcYUC), TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (CcTAA1), and GRETCHEN HAGEN 3 (GH3) genes. On the other hand, most of the IAA compounds decreased during the induction of SE. The results presented in this research suggest that a balance between free IAA and its amide conjugates is necessary to allow the expression of SE-related genes. PMID:24299659

  3. Somatic Embryogenesis in Pinus spp.

    PubMed

    Montalbán, Itziar Aurora; García-Mendiguren, Olatz; Moncaleán, Paloma

    2016-01-01

    Somatic embryogenesis (SE) has been the most important development for plant tissue culture, not only for mass propagation but also for enabling the implementation of biotechnological tools that can be used to increase the productivity and wood quality of plantation forestry. Development of SE in forest trees started in 1985 and nowadays many studies are focused on the optimization of conifer SE system. However, these advances for many Pinus spp. are not sufficiently refined to be implemented commercially. In this chapter, a summary of the main systems used to achieve SE in Pinus spp. is reported. PMID:26619876

  4. Somatic Embryogenesis and Genetic Modification of Vitis.

    PubMed

    Dhekney, Sadanand A; Li, Zhijian T; Grant, Trudi N L; Gray, Dennis J

    2016-01-01

    Grapevine embryogenic cultures are ideal target tissues for inserting desired traits of interest and improving existing cultivars via precision breeding (PB). PB is a new approach that, like conventional breeding, utilizes only DNA fragments obtained from sexually compatible grapevine plants. Embryogenic culture induction occurs by placing leaves or stamens and pistils on induction medium with a dark/light photoperiod cycle for 12-16 weeks. Resulting cultures produce sectors of embryogenic and non-embryogenic callus, which can be identified on the basis of callus morphology and color. Somatic embryo development occurs following transfer of embryogenic callus to development medium and cultures can be maintained for extended periods of time by transfer of the proliferating proembryonic masses to fresh medium at 4-6-week intervals. To demonstrate plant recovery via PB, somatic embryos at the mid-cotyledonary stage are cocultivated with Agrobacterium containing the desired gene of interest along with a, non-PB, enhanced green fluorescent protein/neomycin phosphotransferase II (egfp/nptII) fusion gene. Modified cultures are grown on proliferation and development medium to produce uniformly modified somatic embryos via secondary embryogenesis. Modified embryos identified on the basis of green fluorescence and kanamycin resistance are transferred to germination medium for plant development. The resulting plants are considered to prototype examples of the PB approach, since they contain egfp/nptII, a non-grapevine-derived fusion gene. Uniform green fluorescent protein (GFP) fluorescence can be observed in all tissues of regenerated plants. PMID:26619866

  5. Induction of somatic embryogenesis in explants of shoot cultures established from adult Eucalyptus globulus and E. saligna × E. maidenii trees.

    PubMed

    Corredoira, E; Ballester, A; Ibarra, M; Vieitez, A M

    2015-06-01

    A reproducible procedure for induction of somatic embryogenesis (SE) from adult trees of Eucalyptus globulus Labill. and the hybrid E. saligna Smith × E. maidenii has been developed for the first time. Somatic embryos were obtained from both shoot apex and leaf explants of all three genotypes evaluated, although embryogenic frequencies were significantly influenced by the species/genotype, auxin and explant type. Picloram was more efficient for somatic embryo induction than naphthaleneacetic acid (NAA), with the highest frequency of induction being obtained in Murashige and Skoog medium containing 40 µM picloram and 40 mg l(-1) gum Arabic, in which 64% of the shoot apex explants and 68.8% of the leaf explants yielded somatic embryos. The embryogenic response of the hybrid was higher than that of the E. globulus, especially when NAA was used. The cultures initiated on picloram-containing medium consisted of nodular embryogenic structures surrounded by a mucilaginous coating layer that emerged from a watery callus developed from the initial explants. Cotyledonary somatic embryos were differentiated after subculture of these nodular embryogenic structures on a medium lacking plant growth regulators. Histological analysis confirmed the bipolar organization of the somatic embryos, with shoot and root meristems and closed procambial tissue that bifurcated into small cotyledons. The root pole was more differentiated than the shoot pole, which appeared to be formed by a few meristematic layers. Maintenance of the embryogenic lines by secondary SE was attained by subculturing individual cotyledonary embryos or small clusters of globular and torpedo embryos on medium with 16.11 µM NAA at 4- to 5-week intervals. Somatic embryos converted into plantlets after being transferred to liquid germination medium although plant regeneration remained poor. PMID:25877768

  6. Somatic Embryogenesis of Lilium from Microbulb Transverse Thin Cell Layers.

    PubMed

    Marinangeli, Pablo

    2016-01-01

    A reliable somatic embryogenesis protocol is a prerequisite for application of other plant biotechniques. Several protocols were reported for genus Lilium, with variable success. Between them, transverse Thin Cell Layers (tTCL) were used efficiently to induce indirect somatic embryogenesis of Lilium. Somatic embryogenesis potential is dependent on the genotype, explant, and culture medium composition, especially as for plant growth regulators and environmental conditions. Usually, the process comprises three phases: embryogenic callus induction, embryogenic callus proliferation and somatic embryo germination. Somatic embryo germination can be achieved in light or dark. In the first case, complete plantlets are formed, with green leaves and pseudobulb in the base. In darkness, microbulbs are formed from single somatic embryos or clusters. A last phase of microbulb enlargement allows plantlets or microbulbs to increase their biomass. These enlarged microbulbs do not need special acclimatization conditions when transferred to soil and quickly produce sturdy plants. This chapter describes a protocol for somatic embryogenesis of Lilium using tTCL from microbulbs. PMID:26619874

  7. Somatic Embryogenesis in Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae).

    PubMed

    Guerra, Miguel P; Steiner, Neusa; Farias-Soares, Francine L; Vieira, Leila do N; Fraga, Hugo P F; Rogge-Renner, Gladys D; Maldonado, Sara B

    2016-01-01

    This chapter deals with the features of somatic embryogenesis (SE) in Araucaria angustifolia, an endangered and native conifer from south Brazil. In this species SE includes the induction and proliferation of embryogenic cultures composed of pro-embryogenic masses (PEMs), which precede somatic embryos development. A. angustifolia SE model encompasses induction, proliferation, pre-maturation, and maturation steps. Double-staining with acetocarmine and Evan's blue is useful to evaluate the embryonic somatic structures. In this chapter we describe A. angustifolia SE protocols and analyzes morphological features in the different SE developmental stages. PMID:26619879

  8. Somatic Embryogenesis in Crocus sativus L.

    PubMed

    Sevindik, Basar; Mendi, Yesim Yalcin

    2016-01-01

    Saffron (Crocus sativus L.) is one of the most important species in Crocus genus because of its effective usage. It is not only a very expensive spice, but it has also a big ornamental plant potential. Crocus species are propagated by corm and seed, and male sterility is the most important problem of this species. Hence, somatic embryogenesis can be regarded as a strategic tool for the multiplication of saffron plants. In this chapter, the production of saffron corms via somatic embryogenesis is described. PMID:26619871

  9. Somatic Embryogenesis in Lisianthus (Eustoma russellianum Griseb.).

    PubMed

    Ruffoni, Barbara; Bassolino, Laura

    2016-01-01

    Somatic embryogenesis is, for the main floricultural crops, a promising system for commercial scale-up, providing cloned material to be traded as seedlings. Somatic embryos, having the contemporary presence of root apical meristem and shoot apical meristem, can be readily acclimatized. For Lisianthus it is possible to induce embryogenic callus from leaf fragments of selected genotypes and to obtain embryos either in agarized substrate or in liquid suspension culture. The production of somatic embryos in liquid medium is high and can be modulated in order to synchronize the cycle and the size of the neoformed structures. The possibility to use the liquid substrate with high propagation rates reduces labor costs and could support the costs of eventual automation. In this paper we report a stepwise protocol for somatic embryogenesis in the species Eustoma russellianum. PMID:26619872

  10. Somatic embryogenesis in Hedychium bousigonianum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An efficient primary somatic embryo (SE) and secondary somatic embryo (SSE) production system was developed for the ornamental ginger Hedychium bousigonianum Pierre ex Gagnepain. Addition of two ethylene inhibitors, salicylic acid (SA) and silver nitrate (AgNO3), to the culture media improved the sy...

  11. Somatic Embryogenesis in Peach Palm Using the Thin Cell Layer Technique: Induction, Morpho-histological Aspects and AFLP Analysis of Somaclonal Variation

    PubMed Central

    Steinmacher, D. A.; Krohn, N. G.; Dantas, A. C. M.; Stefenon, V. M.; Clement, C. R.; Guerra, M. P.

    2007-01-01

    Background and Aims The thin cell layer (TCL) technique is based on the use of very small explants and has allowed enhanced in vitro morphogenesis in several plant species. The present study evaluated the TCL technique as a procedure for somatic embryo production and plantlet regeneration of peach palm. Methods TCL explants from different positions in the shoot apex and leaf sheath of peach palm were cultivated in MS culture medium supplemented with 0–600 µm Picloram in the presence of activated charcoal. The production of primary calli and embryogenic calli was evaluated in these different conditions. Histological and amplified fragment length polymorphism (AFLP) analyses were conducted to study in vitro morphogenetic responses and genetic stability, respectively, of the regenerated plantlets. Key Results Abundant primary callus induction was observed from TCLs of the shoot meristem in culture media supplemented with 150–600 µm Picloram (83–97 %, respectively). The production of embryogenic calli depends on Picloram concentration and explant position. The best response observed was 43 % embryogenic callus production from shoot meristem TCL on 300 µm Picloram. In maturation conditions, 34 ± 4 somatic embryos per embryogenic callus were obtained, and 45·0 ± 3·4 % of these fully developed somatic embryos were converted, resulting in plantlets ready for acclimatization, of which 80 % survived. Histological studies revealed that the first cellular division events occurred in cells adjacent to vascular tissue, resulting in primary calli, whose growth was ensured by a meristematic zone. A multicellular origin of the resulting somatic embryos arising from the meristematic zone is suggested. During maturation, histological analyses revealed bipolarization of the somatic embryos, as well as the development of new somatic embryos. AFLP analyses revealed that 92 % of the regenerated plantlets were true to type. The use of TCL explants considerably improves the number of calli and somatic embryos produced in comparison with previously described protocols for in vitro regeneration of peach palm. Conclusions The present study suggests that the TCL somatic embryogenesis protocol developed is feasible, although it still requires further optimization for in vitro multiplication of peach palm, especially the use of similar explants obtained from adult palm trees. PMID:17670751

  12. Potential link between biotic defense activation and recalcitrance to induction of somatic embryogenesis in shoot primordia from adult trees of white spruce (Picea glauca)

    PubMed Central

    2013-01-01

    Background Among the many commercial opportunities afforded by somatic embryogenesis (SE), it is the ability to clonally propagate individual plants with rare or elite traits that has some of the most significant implications. This is particularly true for many long-lived species, such as conifers, but whose long generation times pose substantive challenges, including increased recalcitrance for SE as plants age. Identification of a clonal line of somatic embryo-derived trees whose shoot primordia have remained responsive to SE induction for over a decade, provided a unique opportunity to examine the molecular aspects underpinning SE within shoot tissues of adult white spruce trees. Results Microarray analysis was used to conduct transcriptome-wide expression profiling of shoot explants taken from this responsive genotype following one week of SE induction, which when compared with that of a nonresponsive genotype, led to the identification of four of the most differentially expressed genes within each genotype. Using absolute qPCR to expand the analysis to three weeks of induction revealed that differential expression of all eight candidate genes was maintained to the end of the induction treatment, albeit to differing degrees. Most striking was that both the magnitude and duration of candidate gene expression within the nonresponsive genotype was indicative of an intense physiological response. Examining their putative identities further revealed that all four encoded for proteins with similarity to angiosperm proteins known to play prominent roles in biotic defense, and that their high-level induction over an extended period is consistent with activation of a biotic defense response. In contrast, the more temperate response within the responsive genotype, including induction of a conifer-specific dehydrin, is more consistent with elicitation of an adaptive stress response. Conclusions While additional evidence is required to definitively establish an association between SE responsiveness and a specific physiological response, these results suggest that biotic defense activation may be antagonistic, likely related to the massive transcriptional and metabolic reprogramming that it elicits. A major issue for future work will be to determine how and if suppressing biotic defense activation could be used to promote a physiological state more conducive to SE induction. PMID:23937238

  13. Morphohistological analysis and histochemistry of Feijoa sellowiana somatic embryogenesis.

    PubMed

    Cangahuala-Inocente, G C; Steiner, N; Santos, M; Guerra, M P

    2004-10-01

    Morphohistological analysis and histochemical studies were carried out during the induction and development of Feijoa sellowiana somatic embryos. Zygotic embryos were cultured on LPm medium containing 2,4-dichlorophenoxyacetic acid (20 microM) and glutamine (8 mM). Somatic embryogenesis could be induced from embryogenic cells that originated in meristematic centers or from clusters of cells. The presence of few starch grains and abundant protein bodies was observed in the globular and early torpedo stages, while in torpedo and cotyledonary-stage somatic embryos an enhanced synthesis of starch grains was associated with the accumulation of reserves to be used in the conversion of the embryos to plantlets. Proteins were predominantly observed in protoderm cells, as well as in the meristematic apical region of torpedo and cotyledonary-stage somatic embryos. PMID:15726807

  14. Studies on Somatic Embryogenesis in Sweetpotato

    NASA Technical Reports Server (NTRS)

    Bennett, J. Rasheed; Prakash, C. S.

    1997-01-01

    The purpose of this study was to improve the somatic embryo (SE) system for plant production of sweetpotato Ipomoea batatas L.(Lam)l. Explants isolated from SE-derived sweet potato plants were compared with control (non SE-derived) plants for their competency for SE production. Leaf explants were cultured on Murashige-Skoog (MS) medium with 2,4-dichlorophenoxy acetic acid (0.2 mg/L) and 6-benzylaminopurine (2.5 mg/L) for 2 weeks in darkness and transferred to MS medium with abscisic acid (2.5 Explants isolated from those plants developed through somatic embryo-genesis produced new somatic embryos rapidly and in higher frequency than those isolated from control plants. They also appeared to grow faster in tissue culture than the control plants. Current studies in the laboratory are examining whether plants derived from a cyclical embryogenesis system (five cycles) would have any further positive impact on the rapidity and frequency of somatic embryo development. More detailed studies using electron microscopy are expected to show the point of origin of the embryos and to allow determination of their quality throughout the cyclical process. This study may facilitate improved plant micropropagation, gene transfer and germplasm conservation in sweet potato.

  15. Studies for Somatic Embryogenesis in Sweet Potato

    NASA Technical Reports Server (NTRS)

    Bennett, J. Rasheed; Prakash, C. S.

    1997-01-01

    The purpose of this study was to improve the somatic embryo (SE) system for plant production of sweet potato (Ipomoea batatas L(Lam)). Explants isolated from SE-derived sweet potato plants were compared with control (non SE-derived) plants for their competency for SE production. Leaf explants were cultured on Murashige-Skoog (MS) medium with 2,4-dichlorophenoxy acetic acid (0.2 mg/L) and 6-benzylaminopurine (2.5 mg/L) for 2 weeks in darkness and transferred to MS medium with abscisic acid (2.5 mg/L). Explants isolated from those plants developed through somatic embryogenesis produced new somatic embryos rapidly and in higher frequency than those isolated from control plants They also appeared to grow faster in tissue culture than the control plants. Current studies in the laboratory are examining whether plants derived from a cyclical embryogenesis system (five cycles) would have any further positive impact on the rapidity and frequency of somatic embryo development. More detailed studies using electron microscopy are expected to show the point of origin of the embryos and to allow determination of their quality throughout the cyclical process. This study may facilitate improved plant micropropagation, gene transfer and germplasm conservation in sweet potato.

  16. Spaceflight reduces somatic embryogenesis in orchardgrass (Poaceae)

    NASA Technical Reports Server (NTRS)

    Conger, B. V.; Tomaszewski, Z. Jr; McDaniel, J. K.; Vasilenko, A.

    1998-01-01

    Somatic embryos initiate and develop from single mesophyll cells in in vitro cultured leaf segments of orchard-grass (Dactylis glomerata L.). Segments were plated at time periods ranging from 21 to 0.9 d (21 h) prior to launch on an 11 d spaceflight (STS-64). Using a paired t-test, there was no significant difference in embryogenesis from preplating periods of 14 d and 21 d. However, embryogenesis was reduced by 70% in segments plated 21 h before launch and this treatment was significant at P=0.0001. The initial cell divisions leading to embryo formation would be taking place during flight in this treatment. A higher ratio of anticlinal:periclinal first cell divisions observed in the flight compared to the control tissue suggests that microgravity affects axis determination and embryo polarity at a very early stage. A similar reduction in zygotic embryogenesis would reduce seed formation and have important implications for long-term space flight or colonization where seeds would be needed either for direct consumption or to grow another generation of plants.

  17. In vitro plant regeneration of Aster scaber via somatic embryogenesis.

    PubMed

    Boo, Kyung Hwan; Cao, Dang Viet; Pamplona, Reniel S; Lee, Doseung; Riu, Key-Zung; Lee, Dong-Sun

    2015-01-01

    We established an in vitro plant regeneration system via somatic embryogenesis of Aster scaber, an important source of various biologically active phytochemicals. We examined the callus induction and embryogenic capacities of three explants, including leaves, petioles, and roots, on 25 different media containing different combinations of α-naphthalene acetic acid (NAA) and 6-benzyladenine (BA). The optimum concentrations of NAA and BA for the production of embryogenic calli were 5.0 μM and 0.05 μM, respectively. Media containing higher concentrations of auxin and cytokinin (such as 25 μM NAA and 25 μM BA) were suitable for shoot regeneration, especially for leaf-derived calli, which are the most readily available calli and are highly competent. For root induction from regenerated shoots, supplemental auxin and/or cytokinin did not improve rooting, but instead caused unwanted callus induction or retarded growth of regenerated plants. Therefore, plant growth regulator-free medium was preferable for root induction. Normal plants were successfully obtained from calli under the optimized conditions described above. This is the first report of the complete process of in vitro plant regeneration of A. scaber via somatic embryogenesis. PMID:25640866

  18. Reproduction of the Medicinal Plant Pelargonium sidoides via Somatic Embryogenesis.

    PubMed

    Duchow, Stefanie; Blaschek, Wolfgang; Classen, Birgit

    2015-08-01

    The medicinal plant Pelargonium sidoides DC. (Geraniaceae) was traditionally used for the treatment of the common cold and cough in South Africa. Today an aequous-ethanolic root extract from this plant is approved for the treatment of acute bronchitis and is globally marketed also as an immunostimulant. The increasing demand of the plant material for the industrial production indicates the need of new effective methods for the propagation of P. sidoides. Here we report somatic embryogenesis and in vitro plantlet regeneration from somatic cells of inflorescence shoots and petioles of P. sidoides. A one-week cultivation of explants in media containing different concentrations of thidiazuron (1, 2.2, 3, and 4 mg/L) followed by a cultivation period without phytohormones resulted in the induction of somatic embryos within 2-4 weeks. After 2-4 months, the embryos generated roots and could be transferred into a greenhouse, where flower formation took place and the development of seeds occurred with high germination rates. The root umckalin concentration, determined by high-performance thin-layer chromatography, was comparable to that of seed-cultivated plants (100 ± 6 vs. 113 ± 10 µg umckalin/g dried roots). For the first time, direct somatic embryogenesis has been established as an appropriate cultivation method for P. sidoides plants used as raw material in the pharmaceutical industry. Moreover, genetically identical plants (chemical races) can be easily generated by this procedure. PMID:26287694

  19. Somatic Embryogenesis in Four Tree Legumes

    PubMed Central

    Das, Premananda

    2011-01-01

    Somatic embryogenesis was achieved in four leguminous tree species, that is, Acacia catechu, Acacia arabica, Hardwickia binata, and Dalbergia sissoo using immature zygotic embryos as explants on Murashige and Skoog (MS) medium supplemented with 0.25–1.0 mg/l Kn (kinetin) and 2.0–3.0 mg/l 2,4-D (2,4-dichlorophenoxyacetic acid) or NAA (1-napthaleneacetic acid) and 3% sucrose. MS medium containing 2.0 mg/l 2,4-D and 1.0–1.5 mg/l Kn was noted to be most effective in inducing friable embryogenic callus (FEC); the number of somatic embryos per culture varied in MS medium supplemented with 1.0–2.0 mg/l 2,4-D or NAA and 0.25–1.5 mg/l kinetin. The maximum number of somatic embryos was obtained in MS medium containing 1.5–2.0 mg/l 2,4-D or NAA and 1.0–1.5 mg/l kinetin; proliferation of embryogenic calli was enhanced in cultures having 1.0–2.0 mg/l 2,4-D, 1.0–1.5 mg/l kinetin, and 400–600 mg/l L-Proline. The somatic embryos in various shapes and sizes after the first subculture on MS medium supplemented with 0.1 mg/l IAA and 0.25 mg/l BA; developed shoots and rooted in 1/2 strength MS medium supplemented with 0.1 mg/l IBA or IAA. The somatic embryo-derived plantlets were transferred to the field after being hardened in the climate-controlled hardening chamber. PMID:21350667

  20. Somatic Embryogenesis: Identified Factors that Lead to Embryogenic Repression. A Case of Species of the Same Genus

    PubMed Central

    Nic-Can, Geovanny I.; Galaz-Ávalos, Rosa M.; De-la-Peña, Clelia; Alcazar-Magaña, Armando; Wrobel, Kazimierz; Loyola-Vargas, Víctor M.

    2015-01-01

    Somatic embryogenesis is a powerful biotechnological tool for the mass production of economically important cultivars. Due to the cellular totipotency of plants, somatic cells under appropriate conditions are able to develop a complete functional embryo. During the induction of somatic embryogenesis, there are different factors involved in the success or failure of the somatic embryogenesis response. Among these factors, the origin of the explant, the culture medium and the in vitro environmental conditions have been the most studied. However, the secretion of molecules into the media has not been fully addressed. We found that the somatic embryogenesis of Coffea canephora, a highly direct embryogenic species, is disrupted by the metabolites secreted from C. arabica, a poorly direct embryogenic species. These metabolites also affect DNA methylation. Our results show that the abundance of two major phenolic compounds, caffeine and chlorogenic acid, are responsible for inhibiting somatic embryogenesis in C. canephora. PMID:26038822

  1. Somatic Embryogenesis: Identified Factors that Lead to Embryogenic Repression. A Case of Species of the Same Genus.

    PubMed

    Nic-Can, Geovanny I; Galaz-Ávalos, Rosa M; De-la-Peña, Clelia; Alcazar-Magaña, Armando; Wrobel, Kazimierz; Loyola-Vargas, Víctor M

    2015-01-01

    Somatic embryogenesis is a powerful biotechnological tool for the mass production of economically important cultivars. Due to the cellular totipotency of plants, somatic cells under appropriate conditions are able to develop a complete functional embryo. During the induction of somatic embryogenesis, there are different factors involved in the success or failure of the somatic embryogenesis response. Among these factors, the origin of the explant, the culture medium and the in vitro environmental conditions have been the most studied. However, the secretion of molecules into the media has not been fully addressed. We found that the somatic embryogenesis of Coffea canephora, a highly direct embryogenic species, is disrupted by the metabolites secreted from C. arabica, a poorly direct embryogenic species. These metabolites also affect DNA methylation. Our results show that the abundance of two major phenolic compounds, caffeine and chlorogenic acid, are responsible for inhibiting somatic embryogenesis in C. canephora. PMID:26038822

  2. Somatic Embryogenesis of Abies cephalonica Loud.

    PubMed

    Krajňáková, Jana; Häggman, Hely

    2016-01-01

    Greek fir (Abies cephalonica Loudon) belongs to the Mediterranean fir species and is widely distributed in the mountains of Central and Southern Greece. Considering a climatic scenario, infestation by pathogens or insects and fire episodes, it has been proposed that Mediterranean firs could be in danger in some parts of their present range but, on the other hand, could also replace other species in more northern zones with temperate humid climates (e.g., silver fir, Abies alba Mill.). As fir species are generally highly productive and therefore important for commercial forestry, they have traditionally been involved in conventional tree improvement programs. A lot of effort has been put into the development of vegetative propagation methods for firs, in order to rapidly gain the benefits of traditional breeding to be utilized in reforestation. The present paper provides up to date information on protocols for somatic embryogenesis (i.e., the most promising in vitro method for vegetative propagation) of Greek fir. Moreover, the protocols for cryopreservation and long-term storage of embryogenic material are described as well. PMID:26619877

  3. Somatic Embryogenesis: Still a Relevant Technique in Citrus Improvement.

    PubMed

    Omar, Ahmad A; Dutt, Manjul; Gmitter, Frederick G; Grosser, Jude W

    2016-01-01

    The genus Citrus contains numerous fresh and processed fruit cultivars that are economically important worldwide. New cultivars are needed to battle industry threatening diseases and to create new marketing opportunities. Citrus improvement by conventional methods alone has many limitations that can be overcome by applications of emerging biotechnologies, generally requiring cell to plant regeneration. Many citrus genotypes are amenable to somatic embryogenesis, which became a key regeneration pathway in many experimental approaches to cultivar improvement. This chapter provides a brief history of plant somatic embryogenesis with focus on citrus, followed by a discussion of proven applications in biotechnology-facilitated citrus improvement techniques, such as somatic hybridization, somatic cybridization, genetic transformation, and the exploitation of somaclonal variation. Finally, two important new protocols that feature plant regeneration via somatic embryogenesis are provided: protoplast transformation and Agrobacterium-mediated transformation of embryogenic cell suspension cultures. PMID:26619868

  4. Callose Deposition Is Required for Somatic Embryogenesis in Plasmolyzed Eleutherococcus senticosus Zygotic Embryos

    PubMed Central

    Tao, Lei; Yang, Yang; Wang, Qiuyu; You, Xiangling

    2012-01-01

    Dynamic changes in callose content, which is deposited as a plant defense response to physiological changes, were analyzed during somatic embryogenesis in Eleutherococcus senticosus zygotic embryos plasmolyzed in 1.0 M mannitol. During plasmolysis, callose deposition was clearly observed inside the plasma membrane of zygotic embryo epidermal cells using confocal laser scanning microscopy. The callose content of zygotic embryos gradually increased between 0 and 12 h plasmolysis and remained stable after 24 h plasmolysis. During eight weeks induction of somatic embryogenesis, the callose content of explants plasmolyzed for 12 h was slightly higher than explants plasmolyzed for 6 or 24 h, with the largest differences observed after 6 weeks culture, which coincided with the maximum callose content and highest number of globular somatic embryos. The highest frequency of somatic embryo formation was observed in explants plasmolyzed for 12 h. The somatic embryo induction rate and number of somatic embryos per explant were markedly different in zygotic embryos pretreated with plasmolysis alone (78.0%, 43 embryos per explant) and those pretreated with plasmolysis and the callose synthase inhibitor 2-deoxy-d-glucose (11.5%, 8 embryos per explant). This study indicates that callose production is required for somatic embryogenesis in plasmolyzed explants. PMID:23203053

  5. Somatic embryogenesis and plant regeneration of cassava (Manihot esculenta Crantz) landraces from Cameroon.

    PubMed

    Mongomake, Kone; Doungous, Oumar; Khatabi, Behnam; Fondong, Vincent N

    2015-01-01

    A procedure to regenerate cassava (Manihot esculenta Crantz) cultivars from Cameroon via somatic embryogenesis (SE) was developed. Shoot apical meristems and immature leaf lobes were used as explants on Murashige and Skoog (MS) basal medium containing 33 or 50 µM of the auxins Picloram (Pic), 2,4-Dichlorophenoxyacetic acid (2,4-D), Dicamba (Dic), and α-Naphthalene acetic acid. Cultivar performance was assessed using SE and number of somatic embryos produced. Overall, the frequency of primary somatic embryogenesis (PSE) and the mean number of somatic embryos produced varied considerably with genotype, type of auxin and concentration tested. For example, cultivar (cv.) Ngan Mbada showed the best performance on MS medium supplemented with 50 µM Pic with a SE frequency of 40 % and an average number of somatic embryos of 90. The second best performance was recorded in cv. Local Red on MS medium supplemented with 33 µM 2,4-D, where the SE frequency was 40 % and an average number of somatic embryos of 60.5. Cultivar Ekona Red recorded the best performance on medium supplemented with 50 µM Pic showing a SE frequency of 47 % and an average number of somatic embryos of 45. We further examined secondary and cyclic somatic embryogenesis (SSE, CSE) and both were also observed to vary with genotype, however, both exhibited significantly higher frequencies of SE compared with PSE. SE started to decline at the fourth cycle of embryogenesis. Examination of organogenesis showed that shoot bud induction from green cotyledons varied across cultivars and benzylaminopurine was shown to outperform Thidiazuron in the ability to induce organogenesis. Furthermore, the frequencies of bud induction were identical under light and dark conditions. Finally, regenerated plants grew easily in the greenhouse with 90-100 % survival rate and did not display detectable variation in morphology. PMID:26361578

  6. A temporary immersion system improves in vitro regeneration of peach palm through secondary somatic embryogenesis

    PubMed Central

    Steinmacher, D. A.; Guerra, M. P.; Saare-Surminski, K.; Lieberei, R.

    2011-01-01

    Background and Aims Secondary somatic embryogenesis has been postulated to occur during induction of peach palm somatic embryogenesis. In the present study this morphogenetic pathway is described and a protocol for the establishment of cycling cultures using a temporary immersion system (TIS) is presented. Methods Zygotic embryos were used as explants, and induction of somatic embryogenesis and plantlet growth were compared in TIS and solid culture medium. Light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to describe in vitro morphogenesis and accompany morpho-histological alterations during culture. Key Results The development of secondary somatic embryos occurs early during the induction of primary somatic embryos. Secondary somatic embryos were observed to develop continually in culture, resulting in non-synchronized development of these somatic embryos. Using these somatic embryos as explants allowed development of cycling cultures. Somatic embryos had high embryogenic potential (65·8 ± 3·0 to 86·2 ± 5·0 %) over the period tested. The use of a TIS greatly improved the number of somatic embryos obtained, as well as subsequent plantlet growth. Histological analyses showed that starch accumulation precedes the development of somatic embryos, and that these cells presented high nucleus/cytoplasm ratios and high mitotic indices, as evidenced by DAPI staining. Morphological and SEM observations revealed clusters of somatic embryos on one part of the explants, while other parts grew further, resulting in callus tissue. A multicellular origin of the secondary somatic embryos is hypothesized. Cells in the vicinity of callus accumulated large amounts of phenolic substances in their vacuoles. TEM revealed that these cells are metabolically very active, with the presence of numerous mitochondria and Golgi apparatuses. Light microscopy and TEM of the embryogenic sector revealed cells with numerous amyloplasts, large nuclei and nucleoli, and numerous plasmodesmata. Plantlets were obtained and after 3 months in culture their growth was significantly better in TIS than on solid culture medium. However, during acclimatization the survival rate of TIS-grown plantlets was lower. Conclusions The present study confirms the occurrence of secondary somatic embryos in peach palm and describes a feasible protocol for regeneration of peach palm in vitro. Further optimizations include the use of explants obtained from adult palms and improvement of somatic embryo conversion rates. PMID:21355009

  7. A microdroplet cell culture based high frequency somatic embryogenesis system for pigeonpea, Cajanus cajan (L.) Millsp.

    PubMed

    Kumar, Nagan Udhaya; Gnanaraj, Muniraj; Sindhujaa, Vajravel; Viji, Maluventhen; Manoharan, Kumariah

    2015-09-01

    A protocol for high frequency production of somatic embryos was worked out in pigeonpea, Cajanus cajan (L.) Millsp. The protocol involved sequential employment of embryogenic callus cultures, low density cell suspension cultures and a novel microdroplet cell culture system. The microdroplet cell cultures involved culture of a single cell in 10 ?I of Murashige and Skoog's medium supplemented with phytohormones, growth factors and phospholipid precursors. By employing the microdroplet cell cultures, single cells in isolation were grown into cell clones which developed somatic embryos. Further, 2,4-dichlorophenoxyacetic acid, kinetin, polyethylene glycol, putrescine, spermine, spermidine, choline chloride, ethanolamine and LiCl were supplemented to the low density cell suspension cultures and microdroplet cell cultures to screen for their cell division and somatic embryogenesis activity. Incubation of callus or the inoculum employed for low density cell suspension cultures and microdroplet cell cultures with polyethylene glycol was found critical for induction of somatic embryogenesis. Somatic embryogenesis at a frequency of 1.19, 3.16 and 6.51 per 10(6) cells was achieved in the callus, low density cell suspension cultures and microdroplet cell cultures, respectively. Advantages of employing microdroplet cell cultures for high frequency production of somatic embryos and its application in genetic transformation protocols are discussed. PMID:26548080

  8. Somatic Embryogenesis in Two Orchid Genera (Cymbidium, Dendrobium).

    PubMed

    da Silva, Jaime A Teixeira; Winarto, Budi

    2016-01-01

    The protocorm-like body (PLB) is the de facto somatic embryo in orchids. Here we describe detailed protocols for two orchid genera (hybrid Cymbidium Twilight Moon 'Day Light' and Dendrobium 'Jayakarta', D. 'Gradita 31', and D. 'Zahra FR 62') for generating PLBs. These protocols will most likely have to be tweaked for different cultivars as the response of orchids in vitro tends to be dependent on genotype. In addition to primary somatic embryogenesis, secondary (or repetitive) somatic embryogenesis is also described for both genera. The use of thin cell layers as a sensitive tissue assay is outlined for hybrid Cymbidium while the protocol outlined is suitable for bioreactor culture of D. 'Zahra FR 62'. PMID:26619873

  9. Role of trace elements in somatic embryogenesis A PIXE study

    NASA Astrophysics Data System (ADS)

    Saha, P.; Raychaudhuri, S.; Mishra, D.; Chakraborty, A.; Sudarshan, M.

    2008-03-01

    Proton induced X-ray emission was used to study the trace elemental profiles of embryogenic and non-embryogenic callus of an important cash crop of India - Plantago ovata. Somatic embryogenesis, a well-known process for plant regeneration and crop improvement is modulated by various factors such as ionizing radiation and micro nutrients in the growth media. The present work reports the trace element variation in normal and irradiated callus tissue of P. ovata. Embryogenic and non-embryogenic callus tissues were exposed to gamma rays from a 60Co gamma source. The absorbed dose ranged from 10 to 100 Gy. Subsequent experiments showed significant dose dependent alterations in K, Ca, Mn, Fe, Ni, Cu, Zn, Br, Sr in both the embryogenic and non-embryogenic callus. The precise involvement of these elements has been discussed in light of somatic embryogenesis of the selected medicinal plant.

  10. Alternative oxidase involvement in Daucus carota somatic embryogenesis.

    PubMed

    Frederico, António Miguel; Campos, Maria Doroteia; Cardoso, Hélia Guerra; Imani, Jafargholi; Arnholdt-Schmitt, Birgit

    2009-12-01

    Plant alternative oxidase (AOX) is a mitochondrial inner membrane enzyme involved in alternative respiration. The critical importance of the enzyme during acclimation upon stress of plant cells is not fully understood and is still an issue of intensive research and discussion. Recently, a role of AOX was suggested for the ability of plant cells to change easily its fate upon stress. In order to get new insights about AOX involvement in cell reprogramming, quantitative real-time polymerase chain reaction (PCR) and inhibitor studies were performed during cell redifferentiation and developmental stages of Daucus carota L. somatic embryogenesis. Transcript level analysis shows that D. carota AOX genes (DcAOX1a and DcAOX2a) are differentially expressed during somatic embryogenesis. DcAOX1a shows lower expression levels, being mainly down-regulated, whereas DcAOX2a presented a large up-regulation during initiation of the realization phase of somatic embryogenesis. However, when globular embryos start to develop, both genes are down-regulated, being this state transient for DcAOX2a. In addition, parallel studies were performed using salicylhydroxamic acid (SHAM) in order to inhibit AOX activity during the realization phase of somatic embryogenesis. Embryogenic cells growing in the presence of the inhibitor were unable to develop embryogenic structures and its growth rate was diminished. This effect was reversible and concentration dependent. The results obtained contribute to the hypothesis that AOX activity supports metabolic reorganization as an essential part of cell reprogramming and, thus, enables restructuring and de novo cell differentiation. PMID:19863756

  11. Proteomic analysis of somatic embryogenesis in Vitis vinifera.

    PubMed

    Marsoni, Milena; Bracale, Marcella; Espen, Luca; Prinsi, Bhakti; Negri, Alfredo S; Vannini, Candida

    2008-02-01

    Two dimensional gel electrophoresis coupled to mass spectrometry has been used to study the somatic embryogenesis in Vitis vinifera, by comparing embryogenic and non embryogenic calluses of the Thompson seedless cv. More than 1,000 spots were reproducibly resolved in colloidal Coomassie brilliant blue stained gels over a pI nonlinear range of 3-10 in the first dimension and using homogeneous 12.5% polyacrylamide gels in the second dimension. The expression pattern of 35 spots differed significantly between the two samples. These spots were processed by mass spectrometry analysis and the protein identity was assigned by using both the non-redundant protein and EST databases. Several responsive proteins, some already known to be involved in the somatic embryogenesis process while others, for the first time put into relation with this process, have been described. Moreover, they have been subdivided in functional categories, and their putative role is discussed in terms of their relevance in the somatic embryogenesis process. PMID:17874111

  12. Plant regeneration via somatic embryogenesis and shoot organogenesis from immature cotyledons of Camellia nitidissima Chi.

    PubMed

    Lü, Jinfeng; Chen, Rong; Zhang, Muhan; da Silva, Jaime A Teixeira; Ma, Guohua

    2013-09-01

    Camellia nitidissima Chi (Theaceae) is a world-famous economic and ornamental plant with golden-yellow flowers. It has been classified as one of the rarest and most endangered plants in China. Our objective was to induce somatic embryogenesis, shoot organogenesis and plant regeneration for C. nitidissima. Three types of callus (whitish, reddish and yellowish) were induced from immature cotyledons on improved woody plant medium (WPM) with different plant growth regulators (PGRs). Among the callus, whitish callus was induced by 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and reddish and yellowish callus were induced by strongly active cytokinins, thidiazuron (TDZ) or 6-benzylaminopurine (BAP), singly or combined with weakly active auxin, α-naphthaleneacetic acid (NAA). The embryogenic callus could differentiate into somatic embryos, nodular embryogenic structures (large embryo-like structures) or adventitious shoots depending on the PGR used in WPM. BAP was best for adventitious buds and zeatin was best for somatic embryogenesis while kinetin (Kt) was best for the formation of nodular embryogenic structures. The three regeneration pathways often occurred in the same embryogenic callus clumps. Most shoots (80.0%) developed roots in WPM supplemented with 24.6 μM IBA and 0.3 μM NAA while 47.5% of somatic embryos could germinate directly and develop into plantlets on induction medium supplemented with 0.9 μM BAP and 0.1 μM NAA. The nodular embryogenic structures could be sub-cultured and cyclically developed in one of two differentiation pathways: shoot organogenesis or somatic embryogenesis. Plantlets derived from shoot buds rooted and somatic embryos germinated when transplanted into soil in a greenhouse; 66.7% of plantlets from shoot culture and 78.6% of plantlets from somatic embryos survived after 8 weeks' acclimatization. PMID:23790533

  13. Effect of Salicylic Acid on Somatic Embryogenesis and Plant Regeneration in Hedychium bousigonianum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to induce somatic embryogenesis in Hedychium bousigonianum Pierre ex Gagnepain and assess the influence of salicylic acid (S) on somatic embryogenesis. Somatic embryos and subsequently regenerated plants were successfully obtained 30 days after transfer of embryogenic...

  14. The role of chromatin modifications in somatic embryogenesis in plants

    PubMed Central

    De-la-Peña, Clelia; Nic-Can, Geovanny I.; Galaz-Ávalos, Rosa M.; Avilez-Montalvo, Randy; Loyola-Vargas, Víctor M.

    2015-01-01

    Somatic embryogenesis (SE) is a powerful tool for plant genetic improvement when used in combination with traditional agricultural techniques, and it is also an important technique to understand the different processes that occur during the development of plant embryogenesis. SE onset depends on a complex network of interactions among plant growth regulators, mainly auxins and cytokinins, during the proembryogenic early stages, and ethylene and gibberellic and abscisic acids later in the development of the somatic embryos. These growth regulators control spatial and temporal regulation of multiple genes in order to initiate change in the genetic program of somatic cells, as well as moderating the transition between embryo developmental stages. In recent years, epigenetic mechanisms have emerged as critical factors during SE. Some early reports indicate that auxins and in vitro conditions modify the levels of DNA methylation in embryogenic cells. The changes in DNA methylation patterns are associated with the regulation of several genes involved in SE, such as WUS, BBM1, LEC, and several others. In this review, we highlight the more recent discoveries in the understanding of the role of epigenetic regulation of SE. In addition, we include a survey of different approaches to the study of SE, and new opportunities to focus SE studies. PMID:26347757

  15. Some ethylene biosynthesis and AP2/ERF genes reveal a specific pattern of expression during somatic embryogenesis in Hevea brasiliensis

    PubMed Central

    2012-01-01

    Background Ethylene production and signalling play an important role in somatic embryogenesis, especially for species that are recalcitrant in in vitro culture. The AP2/ERF superfamily has been identified and classified in Hevea brasiliensis. This superfamily includes the ERFs involved in response to ethylene. The relative transcript abundance of ethylene biosynthesis genes and of AP2/ERF genes was analysed during somatic embryogenesis for callus lines with different regeneration potential, in order to identify genes regulated during that process. Results The analysis of relative transcript abundance was carried out by real-time RT-PCR for 142 genes. The transcripts of ERFs from group I, VII and VIII were abundant at all stages of the somatic embryogenesis process. Forty genetic expression markers for callus regeneration capacity were identified. Fourteen markers were found for proliferating calli and 35 markers for calli at the end of the embryogenesis induction phase. Sixteen markers discriminated between normal and abnormal embryos and, lastly, there were 36 markers of conversion into plantlets. A phylogenetic analysis comparing the sequences of the AP2 domains of Hevea and Arabidopsis genes enabled us to predict the function of 13 expression marker genes. Conclusions This first characterization of the AP2/ERF superfamily in Hevea revealed dramatic regulation of the expression of AP2/ERF genes during the somatic embryogenesis process. The gene expression markers of proliferating callus capacity to regenerate plants by somatic embryogenesis should make it possible to predict callus lines suitable to be used for multiplication. Further functional characterization of these markers opens up prospects for discovering specific AP2/ERF functions in the Hevea species for which somatic embryogenesis is difficult. PMID:23268714

  16. Somatic Embryogenesis in Peach-Palm (Bactris gasipaes) Using Different Explant Sources.

    PubMed

    Steinmacher, Douglas A; Heringer, Angelo Schuabb; Jiménez, Víctor M; Quoirin, Marguerite G G; Guerra, Miguel P

    2016-01-01

    Peach palm (Bactris gasipaes Kunth) is a member of the family Arecaceae and is a multipurpose but underutilized species. Nowadays, fruit production for subsistence and local markets, and heart-of-palm production for local, national, and international markets are the most important uses of this plant. Conventional breeding programs in peach palm are long-term efforts due to the prolonged generation time, large plant size, difficulties with controlled pollination and other factors. Although it is a caespitose palm, its propagation is currently based on seeds, as off-shoots are difficult to root. Hence, tissue culture techniques are considered to be the most likely strategy for efficient clonal plantlet regeneration of this species. Among various techniques, somatic embryogenesis offers the advantages of potential automated large-scale production and putative genetic stability of the regenerated plantlets. The induction of somatic embryogenesis in peach palm can be achieved by using different explant sources including zygotic embryos, immature inflorescences and thin cell layers from the young leaves and shoot meristems. The choice of a particular explant depends on whether clonal propagation is desired or not, as well as on the plant conditions and availability of explants. Protocols to induce and express somatic embryogenesis from different peach palm explants, up to acclimatization of plantlets, are described in this chapter. PMID:26619867

  17. Decoding regulatory landscape of somatic embryogenesis reveals differential regulatory networks between japonica and indica rice subspecies.

    PubMed

    Indoliya, Yuvraj; Tiwari, Poonam; Chauhan, Abhisekh Singh; Goel, Ridhi; Shri, Manju; Bag, Sumit Kumar; Chakrabarty, Debasis

    2016-01-01

    Somatic embryogenesis is a unique process in plants and has considerable interest for biotechnological application. Compare to japonica, indica rice has been less responsive to in vitro culture. We used Illumina Hiseq 2000 sequencing platform for comparative transcriptome analysis between two rice subspecies at six different developmental stages combined with a tag-based digital gene expression profiling. Global gene expression among different samples showed greater complexity in japonica rice compared to indica which may be due to polyphyletic origin of two rice subspecies. Expression pattern in initial stage indicate major differences in proembryogenic callus induction phase that may serve as key regulator to observe differences between both subspecies. Our data suggests that phytohormone signaling pathways consist of elaborate networks with frequent crosstalk, thereby allowing plants to regulate somatic embryogenesis pathway. However, this crosstalk varies between the two rice subspecies. Down regulation of positive regulators of meristem development (i.e. KNOX, OsARF5) and up regulation of its counterparts (OsRRs, MYB, GA20ox1/GA3ox2) in japonica may be responsible for its better regeneration and differentiation of somatic embryos. Comprehensive gene expression information in the present experiment may also facilitate to understand the monocot specific meristem regulation for dedifferentiation of somatic cell to embryogenic cells. PMID:26973288

  18. Decoding regulatory landscape of somatic embryogenesis reveals differential regulatory networks between japonica and indica rice subspecies

    PubMed Central

    Indoliya, Yuvraj; Tiwari, Poonam; Chauhan, Abhisekh Singh; Goel, Ridhi; Shri, Manju; Bag, Sumit Kumar; Chakrabarty, Debasis

    2016-01-01

    Somatic embryogenesis is a unique process in plants and has considerable interest for biotechnological application. Compare to japonica, indica rice has been less responsive to in vitro culture. We used Illumina Hiseq 2000 sequencing platform for comparative transcriptome analysis between two rice subspecies at six different developmental stages combined with a tag-based digital gene expression profiling. Global gene expression among different samples showed greater complexity in japonica rice compared to indica which may be due to polyphyletic origin of two rice subspecies. Expression pattern in initial stage indicate major differences in proembryogenic callus induction phase that may serve as key regulator to observe differences between both subspecies. Our data suggests that phytohormone signaling pathways consist of elaborate networks with frequent crosstalk, thereby allowing plants to regulate somatic embryogenesis pathway. However, this crosstalk varies between the two rice subspecies. Down regulation of positive regulators of meristem development (i.e. KNOX, OsARF5) and up regulation of its counterparts (OsRRs, MYB, GA20ox1/GA3ox2) in japonica may be responsible for its better regeneration and differentiation of somatic embryos. Comprehensive gene expression information in the present experiment may also facilitate to understand the monocot specific meristem regulation for dedifferentiation of somatic cell to embryogenic cells. PMID:26973288

  19. Somatic Embryogenesis and Plant Regeneration of Brachiaria brizantha.

    PubMed

    Cabral, Glaucia B; Carneiro, Vera T C; Dusi, Diva M A; Martinelli, Adriana P

    2016-01-01

    The genus Brachiaria (Trin.) Griseb. belongs to the family Poaceae, order Poales, class Monocotyledonae. In Brachiaria brizantha (Hochst. ex A. Rich.) Stapf., embryogenic callus can be induced from seeds from apomictic plants, which results in high frequency somatic embryo development and plant regeneration. We report here a detailed protocol for callus induction from apomictic seed; followed by in vitro morphogenesis (somatic embryo and bud differentiation), plant regeneration, and acclimatization in the greenhouse. Important details regarding the positioning of seeds for callus induction and precautions to avoid endophytic contamination and the occurrence of albino plants are presented. PMID:26619875

  20. Somatic Embryogenesis and Plant Regeneration in Sapindus mukorossi Gaertn. from Leaf-Derived Callus Induced with 6-Benzylaminopurine.

    PubMed

    Singh, Reetika; Rai, Manoj Kumar; Kumari, Nishi

    2015-09-01

    A somatic embryogenesis system was developed for Sapindus mukorossi Gaertn. from leaf explants obtained from fresh flushes of a mature tree. Callus was induced from the midrib region of leaf explants on Murashige and Skoog (MS) medium containing different concentrations of 2,4-dichlorophenoxyacetic acid or 6-benzylaminopurine. Callus induction and somatic embryogenesis was significantly influenced by the size, physiological age, and orientation of leaf explants on the culture medium and plant growth regulators. Adaxial-side-up orientation of leaf explants significantly promoted embryogenesis in comparison with abaxial-side-up orientation. Maximum number of somatic embryos was induced on MS medium supplemented with 8.88 μM 6-benzylaminopurine. Scanning electron microscopy of embryogenic callus revealed somatic embryo origin and the development of globular-, heart-, and cotyledonary-stage somatic embryos. The frequency of maturation as well as germination of somatic embryos was higher on MS medium containing 8.88 μM 6-benzylaminopurine than on medium without 6-benzylaminopurine. Plantlets which developed from somatic embryos were acclimatized successfully with 90 % survival. PMID:26208689

  1. Label-Free Quantitative Proteomics of Embryogenic and Non-Embryogenic Callus during Sugarcane Somatic Embryogenesis.

    PubMed

    Heringer, Angelo Schuabb; Barroso, Tatiana; Macedo, Amanda Ferreira; Santa-Catarina, Claudete; Souza, Gustavo Henrique Martins Ferreira; Floh, Eny Iochevet Segal; de Souza-Filho, Gonçalo Apolinário; Silveira, Vanildo

    2015-01-01

    The development of somatic cells in to embryogenic cells occurs in several stages and ends in somatic embryo formation, though most of these biochemical and molecular changes have yet to be elucidated. Somatic embryogenesis coupled with genetic transformation could be a biotechnological tool to improve potential crop yields potential in sugarcane cultivars. The objective of this study was to observe somatic embryo development and to identify differentially expressed proteins in embryogenic (E) and non-embryogenic (NE) callus during maturation treatment. E and NE callus were cultured on maturation culture medium supplemented with different concentrations (0.0, 0.75, 1.5 and 2.0 g L(-1)) of activated charcoal (AC). Somatic embryo formation and differential protein expression were evaluated at days 0 and 21 using shotgun proteomic analyses. Treatment with 1.5 g L(-1) AC resulted in higher somatic embryo maturation rates (158 somatic embryos in 14 days) in E callus but has no effect in NE callus. A total of 752 co-expressed proteins were identified through the SUCEST (The Sugarcane EST Project), including many housekeeping proteins. E callus showed 65 exclusive proteins on day 0, including dehydrogenase, desiccation-related protein, callose synthase 1 and nitric oxide synthase. After 21 days on maturation treatment, 14 exclusive proteins were identified in E callus, including catalase and secreted protein. NE callus showed 23 exclusive proteins on day 0 and 10 exclusive proteins after 21 days on maturation treatment, including many proteins related to protein degradation. The induction of maturation leads to somatic embryo development, which likely depends on the expression of specific proteins throughout the process, as seen in E callus under maturation treatment. On the other hand, some exclusive proteins can also specifically prevent of somatic embryos development, as seen in the NE callus. PMID:26035435

  2. Label-Free Quantitative Proteomics of Embryogenic and Non-Embryogenic Callus during Sugarcane Somatic Embryogenesis

    PubMed Central

    Heringer, Angelo Schuabb; Barroso, Tatiana; Macedo, Amanda Ferreira; Santa-Catarina, Claudete; Souza, Gustavo Henrique Martins Ferreira; Floh, Eny Iochevet Segal; de Souza-Filho, Gonçalo Apolinário; Silveira, Vanildo

    2015-01-01

    The development of somatic cells in to embryogenic cells occurs in several stages and ends in somatic embryo formation, though most of these biochemical and molecular changes have yet to be elucidated. Somatic embryogenesis coupled with genetic transformation could be a biotechnological tool to improve potential crop yields potential in sugarcane cultivars. The objective of this study was to observe somatic embryo development and to identify differentially expressed proteins in embryogenic (E) and non-embryogenic (NE) callus during maturation treatment. E and NE callus were cultured on maturation culture medium supplemented with different concentrations (0.0, 0.75, 1.5 and 2.0 g L-1) of activated charcoal (AC). Somatic embryo formation and differential protein expression were evaluated at days 0 and 21 using shotgun proteomic analyses. Treatment with 1.5 g L-1 AC resulted in higher somatic embryo maturation rates (158 somatic embryos in 14 days) in E callus but has no effect in NE callus. A total of 752 co-expressed proteins were identified through the SUCEST (The Sugarcane EST Project), including many housekeeping proteins. E callus showed 65 exclusive proteins on day 0, including dehydrogenase, desiccation-related protein, callose synthase 1 and nitric oxide synthase. After 21 days on maturation treatment, 14 exclusive proteins were identified in E callus, including catalase and secreted protein. NE callus showed 23 exclusive proteins on day 0 and 10 exclusive proteins after 21 days on maturation treatment, including many proteins related to protein degradation. The induction of maturation leads to somatic embryo development, which likely depends on the expression of specific proteins throughout the process, as seen in E callus under maturation treatment. On the other hand, some exclusive proteins can also specifically prevent of somatic embryos development, as seen in the NE callus. PMID:26035435

  3. Somatic Embryogenesis in Horse Chestnut (Aesculus hippocastanum L.).

    PubMed

    Capuana, Maurizio

    2016-01-01

    Embryogenic cultures of horse chestnut (Aesculus hippocastanum L.) can be obtained from different organs and tissues. We describe here the induction from stamen filaments and the procedures applied for the successive phases of somatic embryo development and maturation. Embryogenic tissues are obtained on Murashige and Skoog medium containing 9.0 μM 2,4-dichlorophenoxyacetic acid. Somatic embryos develop after transfer to hormone-free medium enriched with glutamine. Maturation and germination of isolated embryos are achieved by transfer to medium containing polyethylene glycol 4000 and activated charcoal, successive desiccation treatment, and cold storage at 4 °C for 8 weeks. PMID:26619878

  4. Micropropagation of Citrus spp. by organogenesis and somatic embryogenesis.

    PubMed

    Chiancone, Benedetta; Germanà, Maria Antonietta

    2013-01-01

    Citrus spp., the largest fruit crops produced worldwide, are usually asexually propagated by cuttings or grafting onto seedling rootstocks. Most of Citrus genotypes are characterized by polyembryony due to the occurrence of adventive nucellar embryos, which lead to the production of true-to-type plants by seed germination. Tissue culture and micropropagation, in particular, are valuable alternatives to traditional propagation to obtain a high number of uniform and healthy plants in a short time and in a small space. Moreover, in vitro propagation provides a rapid system to multiply the progeny obtained by breeding programs, allows the use of monoembryonic and seedless genotypes as rootstocks, and it is very useful also for breeding and germplasm preservation.In this chapter, two protocols regarding organogenesis of a rootstock and somatic embryogenesis of a cultivar have been described. PMID:23179693

  5. Stress-induced somatic embryogenesis in vegetative tissues of Arabidopsis thaliana.

    PubMed

    Ikeda-Iwai, Miho; Umehara, Mikihisa; Satoh, Shinobu; Kamada, Hiroshi

    2003-04-01

    Somatic embryogenesis is an obvious experimental evidence of totipotency, and is used as a model system for studying the mechanisms of de-differentiation and re-differentiation of plant cells. Although Arabidopsis is widely used as a model plant for genetic and molecular biological studies, there is no available tissue culture system for inducing somatic embryogenesis from somatic cells in this plant. We established a new tissue culture system using stress treatment to induce somatic embryogenesis in Arabidopsis. In this system, stress treatment induced formation of somatic embryos from shoot-apical-tip and floral-bud explants. The somatic embryos grew into young plantlets with normal morphology, including cotyledons, hypocotyls, and roots, and some embryo-specific genes (ABI3 and FUS3) were expressed in these embryos. Several stresses (osmotic, heavy metal ion, and dehydration stress) induced somatic embryogenesis, but the optimum stress treatment differed between different stressors. When we used mannitol to cause osmotic stress, the optimal conditions for somatic embryogenesis were 6-9 h of culture on solid B5 medium containing 0.7 m mannitol, after which the explants were transferred to B5 medium containing 2,4-dichlorophenoxyacetic acid (2,4-D, 4.5 microm), but no mannitol. Using this tissue culture system, we induced somatic embryogenesis in three major ecotypes of Arabidopsis thaliana-Ws, Col, and Ler. PMID:12662313

  6. In vitro somatic embryogenesis and plantlet regeneration from immature male inflorescence of adult dura and tenera palms of Elaeis guineensis (Jacq.).

    PubMed

    Jayanthi, Madhavan; Susanthi, Bollarapu; Murali Mohan, Nandiganti; Mandal, Pranab Kumar

    2015-01-01

    We report here a method for plant regeneration through somatic embryogenesis from explants collected from immature male inflorescence of adult oil palm cultivated in India. Callus induction was successful from tissues of immature male inflorescence collected from both dura and tenera varieties of oil palm. A modified Y3 (Eeuwens) media supplemented with several additives and activated charcoal (3%) were used for the experiments. Out of four different auxin treatments, 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid (picloram) produced maximum callus induction (82%) and it was not significantly different from 2,4-dichlorophenoxyacetic acid (2,4-D) and a combination of 2,4-D + picloram. The callus induction obtained with auxin α-naphthalene acetic acid was only 54% and it was significantly low as compared to the other treatments. Highest embryogenesis was obtained with a combination of 2,4-D + picloram (4.9%) followed by picloram (3.4%). Genotypic variation in response to the same auxins was observed both for callus induction and embryogenesis. Callus induction and embryogenesis ranged from 42 to 72% and 6.8 to 9.35%, respectively in tenera. The formation of embryogenic calli was marked by the appearance of white to yellowish globular or nodular structures which subsequently formed clear somatic embryos. Somatic embryogenesis was asynchronous and at one time we could find different stages of embryogenesis like the globular, torpedo and the cotyledonary stages. The somatic embryos when exposed to light in the same basal media along with 6-benzyladenine (18 µM), abscisic acid (3.78 µM) and gibberellic acid (5.78 µM) regenerated into plantlets. To the best of our knowledge this is the first report o f callus induction and somatic embryogenesis from immature male inflorescence of oil palm. PMID:26085976

  7. Proteomic Analysis of Immature Fraxinus mandshurica Cotyledon Tissues during Somatic Embryogenesis: Effects of Explant Browning on Somatic Embryogenesis

    PubMed Central

    Liu, Chun-Ping; Yang, Ling; Shen, Hai-Long

    2015-01-01

    Manchurian ash (Fraxinus mandshurica Rupr.) is a valuable hardwood species in Northeast China. In cultures of F. mandshurica, somatic embryos were produced mainly on browned explants. Therefore, we studied the mechanism of explant browning and its relationship with somatic embryogenesis (SE). We used explants derived from F. mandshurica immature zygotic embryo cotyledons as materials. Proteins were extracted from browned embryogenic explants, browned non-embryogenic explants, and non-brown explants, and then separated by 2-dimensional electrophoresis. Differentially and specifically expressed proteins were analyzed by mass spectrometry to identify proteins involved in the browning of explants and SE. Some stress response and defense proteins such as chitinases, peroxidases, aspartic proteinases, and an osmotin-like protein played important roles during SE of F. mandshurica. Our results indicated that explant browning might not be caused by the accumulation and oxidation of polyphenols only, but also by some stress-related processes, which were involved in programmed cell death (PCD), and then induced SE. PMID:26084048

  8. An efficient in vitro system for somatic embryogenesis and podophyllotoxin production in Podophyllum hexandrum Royle.

    PubMed

    Rajesh, Manoharan; Sivanandhan, Ganeshan; Jeyaraj, Murugaraj; Chackravarthy, Rajan; Manickavasagam, Markandan; Selvaraj, N; Ganapathi, Andy

    2014-09-01

    Podophyllum hexandrum Royle known as Indian mayapple is an important medicinal plant found only in higher altitudes (2,700 to 4,200 m) of the Himalayas. The highly valued anticancer drug Podophyllotoxin is obtained from the roots of this plant. Due to over exploitation, this endemic plant species is on the verge of extinction. In vitro culture for efficient regeneration and the production of podophyllotoxin is an important research priority for this plant. Hence, in the present study, an efficient plant regeneration system for mass multiplication through somatic embryogenesis was developed. We have screened P. hexandrum seeds collected from three different regions in the Himalayas to find their regenerative potentials. These variants showed variation in germination percentage as well as somatic embryogenic frequency. The seeds collected from the Milam area of Pithoragarh district showed better germination response (99.3%) on Murashige and Skoog (MS) medium fortified with Gibberellic acid (GA3 [5 mg/l]) and higher direct somatic embryogenic frequency (89.6%). Maximum production of embryogenic callus (1.2 g fresh weight [FW]) was obtained when cotyledons containing the direct somatic embryo clusters were cultured in MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D [1.5 mg/l]) after 4 week of culture in complete darkness. In the present investigation, somatic embryogenesis was accomplished either by direct organogenesis or callus mediated pathways. The latter method resulted in a higher frequency of somatic embryo induction in hormone-free MS medium yielding 47.7 embryos/50 mg of embryogenic callus and subsequent germination in MS medium supplemented with GA3 (5 mg/l). Seventy-nine percent of embryos attained complete maturity and germinated into normal plants with well-developed roots. Systematic histological analysis revealed the origin of somatic embryo and their ontogenesis. The higher level of podophyllotoxin (1.8 mg/g dry weight [DW]) was recorded in germinated somatic embryos when compared to field grown plants. The present system can be widely used for mass propagation, transgenic recovery, and podophyllotoxin production for commercial utilization. PMID:24633328

  9. High frequency regeneration via direct somatic embryogenesis and efficient Agrobacterium-mediated genetic transformation of tobacco.

    PubMed

    Pathi, Krishna Mohan; Tula, Suresh; Tuteja, Narendra

    2013-06-01

    A direct somatic embryogenesis protocol was developed for four cultivars of Nicotiana species, by using leaf disc as an explant. Direct somatic embryogenesis of Nicotiana by using BAP and IAA has not been investigated so far. This method does not require formation of callus tissues which leads to somaclonal variations. The frequency of somatic embryogenesis was strongly influenced by the plant growth hormones. The somatic embryos developing directly from explant tissue were noticed after 6 d of culture. Somatic embryogenesis of a high frequency (87-96%) was observed in cultures of the all four genotypes (Nicotiana tabacum, N. benthamiyana, N. xanthi, N. t cv petihavana). The results showed that the best medium for direct somatic embryogenesis was MS supplemented with 2.5 mg/l, 0.2 mg/l IAA and 2% sucrose. Subculture of somatic embryos onto hormone free MS medium resulted in their conversion into plants for all genotypes. About 95% of the regenerated somatic embryos germinated into complete plantlets. The plants showed morphological and growth characteristics similar to those of seed-derived plants. Explants were transformed using Agrobacterium tumifacious LBA4404 plasmid pCAMBIA1301 harboring the GUS gene. The regenerated transgenic plants were confirmed by PCR analysis and histochemical GUS assay. The transformation efficiency obtained by using the Agrobacterium- mediated transformation was more than 95%. This method takes 6 wk to accomplish complete transgenic plants through direct somatic embryogenesis. The transgenic plantlets were acclimatized successfully with 98% survival in greenhouse and they showed normal morphological characteristics and were fertile. The regeneration and transformation method described herein is very simple, highly efficient and fast for the introduction of any foreign gene directly in tobacco through direct somatic embryogenesis. PMID:23518589

  10. High efficiency plant production of North American ginseng via somatic embryogenesis from cotyledon explants.

    PubMed

    Zhou, Sijun; Brown, Daniel C W

    2006-03-01

    An efficient in vitro protocol for plant production of North American ginseng has been established. The pretreatment of cotyledon explants with 1.0 M sucrose at 4 degrees C resulted in an improvement of embryo quality and, combined with a higher sucrose content (7%) in induction medium, improved the embryogenesis frequency from 40% to 75% and the number of embryos per explant from 10 to 21. The frequency of secondary embryogenesis from somatic embryo-derived tissues cultured on MS medium with 1.0 mg l(-1) 2, 4-D and 1.0 mg l(-1) NAA is up to 90%. Somatic embryos can further develop to maturity on SH medium supplemented with 1% activated charcoal and half of them can germinate. About 85% of the germinated embryos will convert into plants with well-developed taproot systems on 1/2 SH medium with 0.5% activated charcoal. The growth chamber and field establishment rates were 95.6 and 93.7%, respectively. The plants transplanted to growth chambers and field plots appear normal. PMID:16283406

  11. Characterization of expression dynamics of WOX homeodomain transcription factors during somatic embryogenesis in Vitis vinifera.

    PubMed

    Gambino, Giorgio; Minuto, Martina; Boccacci, Paolo; Perrone, Irene; Vallania, Rosalina; Gribaudo, Ivana

    2011-01-01

    Different cultivars of Vitis vinifera vary in their potential to form embryogenic tissues. The WUSCHEL (WUS)-related homeobox (WOX) genes have been shown to play an important role in coordinating the gene transcription involved in the early phases of embryogenesis. The expression dynamics of 12 VvWOX genes present in the V. vinifera genome in embryogenic and other tissues of 'Chardonnay' were analysed. In order to understand the influence of WOX genes on the somatic embryogenic process, their expression profiles were compared in two cultivars of V. vinifera ('Chardonnay' and 'Cabernet Sauvignon') that show different aptitudes for embryogenesis. The expression of all VvWOX genes was influenced by culture conditions. VvWOX2 and VvWOX9 were the principal WOX genes expressed during the somatic embryogenesis process, and the low aptitude for embryogenesis of 'Cabernet Sauvignon' was generally correlated with the low expression levels of these VvWOX genes. VvWOX3 and VvWOX11 were strongly activated in correspondence to torpedo and cotyledonary stages of somatic embryos, with low expression in the earlier developmental stages (pre-embryogenic masses and globular embryos) and during embryo germination. VvWOX genes appeared to be key regulators of somatic embryogenesis in grapevine, and the regulation of these genes during early phases of somatic embryogenesis differed between the two cultivars of the same species. PMID:21127025

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

    PubMed

    Nair, R Ramakrishnan; Dutta Gupta, S

    2006-01-01

    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. PMID:16249871

  13. Moderate stress responses and specific changes in polyamine metabolism characterize Scots pine somatic embryogenesis.

    PubMed

    Salo, Heikki M; Sarjala, Tytti; Jokela, Anne; Häggman, Hely; Vuosku, Jaana

    2016-03-01

    Somatic embryogenesis (SE) is one of the methods with the highest potential for the vegetative propagation of commercially important coniferous species. However, many conifers, including Scots pine (Pinus sylvestris L.), are recalcitrant to SE and a better understanding of the mechanisms behind the SE process is needed. In Scots pine SE cultures, embryo production is commonly induced by the removal of auxin, addition of abscisic acid (ABA) and the desiccation of cell masses by polyethylene glycol (PEG). In the present study, we focus on the possible link between the induction of somatic embryo formation and cellular stress responses such as hydrogen peroxide protection, DNA repair, changes in polyamine (PA) metabolism and autophagy. Cellular PA contents and the expression of the PA metabolism genes arginine decarboxylase (ADC), spermidine synthase (SPDS), thermospermine synthase (ACL5) and diamine oxidase (DAO) were analyzed, as well as the expression of catalase (CAT), DNA repair genes (RAD51, KU80) and autophagy-related genes (ATG5, ATG8) throughout the induction of somatic embryo formation in Scots pine SE cultures. Among the embryo-producing SE lines, the expression of ADC, SPDS, ACL5, DAO, CAT, RAD51, KU80 and ATG8 showed consistent profiles. Furthermore, the overall low expression of the stress-related genes suggests that cells in those SE lines were not stressed but recognized the ABA + PEG treatment as a signal to trigger the embryogenic pathway. In those SE lines that were unable to produce embryos, cells seemed to experience the ABA + PEG treatment mostly as osmotic stress and activated a wide range of stress defense mechanisms. Altogether, our results suggest that the direction to the embryogenic pathway is connected with cellular stress responses in Scots pine SE cultures. Thus, the manipulation of stress response pathways may provide a way to enhance somatic embryo production in recalcitrant Scots pine SE lines. PMID:26786537

  14. Extensive Modulation of the Transcription Factor Transcriptome during Somatic Embryogenesis in Arabidopsis thaliana

    PubMed Central

    Gliwicka, Marta; Nowak, Katarzyna; Balazadeh, Salma; Mueller-Roeber, Bernd; Gaj, Malgorzata D.

    2013-01-01

    Molecular mechanisms controlling plant totipotency are largely unknown and studies on somatic embryogenesis (SE), the process through which already differentiated cells reverse their developmental program and become embryogenic, provide a unique means for deciphering molecular mechanisms controlling developmental plasticity of somatic cells. Among various factors essential for embryogenic transition of somatic cells transcription factors (TFs), crucial regulators of genetic programs, are believed to play a central role. Herein, we used quantitative real-time polymerase chain reaction (qRT-PCR) to identify TF genes affected during SE induced by in vitro culture in Arabidopsis thaliana. Expression profiles of 1,880 TFs were evaluated in the highly embryogenic Col-0 accession and the non-embryogenic tanmei/emb2757 mutant. Our study revealed 729 TFs whose expression changes during the 10-days incubation period of SE; 141 TFs displayed distinct differences in expression patterns in embryogenic versus non-embryogenic cultures. The embryo-induction stage of SE occurring during the first 5 days of culture was associated with a robust and dramatic change of the TF transcriptome characterized by the drastic up-regulation of the expression of a great majority (over 80%) of the TFs active during embryogenic culture. In contrast to SE induction, the advanced stage of embryo formation showed attenuation and stabilization of transcript levels of many TFs. In total, 519 of the SE-modulated TFs were functionally annotated and transcripts related with plant development, phytohormones and stress responses were found to be most abundant. The involvement of selected TFs in SE was verified using T-DNA insertion lines and a significantly reduced embryogenic response was found for the majority of them. This study provides comprehensive data focused on the expression of TF genes during SE and suggests directions for further research on functional genomics of SE. PMID:23874927

  15. Interruption of Somatic Embryogenesis in Daucus carota L. by 5-Bromodeoxyuridine

    PubMed Central

    Thomas, John C.; Nessler, Craig; Katterman, Frank

    1989-01-01

    Embryogenic Daucus carota L. cells grown in 9 micromolar 2.4-dichlorophenoxyacetic acid are resistant to greater than 5 micromolar 5-bromodeoxyuridine (BrdU). In contrast, 5 micromolar BrdU strongly inhibits somatic embryogenesis within 24 hours after transfer of cells to an auxin-free medium. DNA synthesis rates in control and BrdU-treated cultures are rapid and similar; however, the DNA content does not reach levels as great in the presence of BrdU as in control cultures. BrdU substitutes for thymidine in the DNA in 28% of the available sites 48 hours after auxin removal. Following DNA repair, somatic embryogenesis resumes. BrdU DNA incorporation leads to somatic embryogenesis inhibition and provides an alternative to auxin treatment for the interruption of carrot cell culture differentiation. Images Figure 2 Figure 3 Figure 7 PMID:16666898

  16. From Stress to Embryos: Some of the Problems for Induction and Maturation of Somatic Embryos.

    PubMed

    Ochatt, Sergio J; Revilla, Maria Angeles

    2016-01-01

    Although somatic embryogenesis has been successfully achieved in numerous plant species, little is known about the mechanism(s) underlying this process. Changes in the balance of growth regulators of the culture medium, osmolarity, or amino acids as well as the genotype and developmental stage of the tissue used as initial explant may have a pivotal influence on the induction of somatic embryogenic cultures. Moreover, different stress agents (ethylene, activated charcoal, cold or heat or electrical shocks), as well as abscisic acid, can also foster the induction or further development of somatic embryos. In the process, cells first return to a stem cell-like status and then either enter their new program or dye when the stress level exceeds cell tolerance. Recalcitrance to differentiation of somatic cells into embryos is frequently observed, and problems such as secondary or recurrent embryogenesis, embryo growth arrest (at the globular stage or during the transition from torpedo to cotyledonary stage), and development of only the aerial part of somatic embryos can appear, interfering with normal germination and conversion of embryos to plants. Some solutions to solve these problems associated to embryogenesis are proposed and two very efficient somatic embryogenesis protocols for two model plant species are detailed. PMID:26619886

  17. Somatic embryogenesis in Solanum tuberosum L.: a histological examination of key developmental stages.

    PubMed

    Sharma, Sanjeev Kumar; Millam, Steve

    2004-09-01

    A potential novel method of producing high-quality potato ( Solanum tuberosum L.) nuclear seeds is through the process of somatic embryogenesis (SE). Somatic embryo formation has been successfully reported in many plant species, but in potato, reliable SE systems are still at the experimental stage. A key factor in the success of any SE system is the ability to discriminate SE-specific cellular structures from those emerging through an organogenic route. In the investigation reported here we attempted to discriminate the progression of specific stages of potato SE by histological means. Internodal segment (INS) explants from 4- to 6-week-old cv. Desiree in vitro cultures were successively cultured on SE induction (for 2 weeks) and expression/regeneration media (for 3 weeks) with and without 2,4-dichlorophenoxyacetic acid (5 microM). Microscopic examination of histological slides prepared using INS explants at different stages revealed the presence of characteristic globular, heart and torpedo stages in the potato SE system along with other associated unique features such as protoderm development and discrete vascular connections. These results confirm the occurrence of potato SE as per the accepted definition of the term. PMID:15185123

  18. Whole transcriptome profiling of maize during early somatic embryogenesis reveals altered expression of stress factors and embryogenesis-related genes.

    PubMed

    Salvo, Stella A G D; Hirsch, Candice N; Buell, C Robin; Kaeppler, Shawn M; Kaeppler, Heidi F

    2014-01-01

    Embryogenic tissue culture systems are utilized in propagation and genetic engineering of crop plants, but applications are limited by genotype-dependent culture response. To date, few genes necessary for embryogenic callus formation have been identified or characterized. The goal of this research was to enhance our understanding of gene expression during maize embryogenic tissue culture initiation. In this study, we highlight the expression of candidate genes that have been previously regarded in the literature as having important roles in somatic embryogenesis. We utilized RNA based sequencing (RNA-seq) to characterize the transcriptome of immature embryo explants of the highly embryogenic and regenerable maize genotype A188 at 0, 24, 36, 48, and 72 hours after placement of explants on tissue culture initiation medium. Genes annotated as functioning in stress response, such as glutathione-S-transferases and germin-like proteins, and genes involved with hormone transport, such as PINFORMED, increased in expression over 8-fold in the study. Maize genes with high sequence similarity to genes previously described in the initiation of embryogenic cultures, such as transcription factors BABY BOOM, LEAFY COTYLEDON, and AGAMOUS, and important receptor-like kinases such as SOMATIC EMBRYOGENESIS RECEPTOR LIKE KINASES and CLAVATA, were also expressed in this time course study. By combining results from whole genome transcriptome analysis with an in depth review of key genes that play a role in the onset of embryogenesis, we propose a model of coordinated expression of somatic embryogenesis-related genes, providing an improved understanding of genomic factors involved in the early steps of embryogenic culture initiation in maize and other plant species. PMID:25356773

  19. Somatic embryogenesis, scanning electron microscopy, histology and biochemical analysis at different developing stages of embryogenesis in six date palm (Phoenix dactylifera L.) cultivars.

    PubMed

    Aslam, Junaid; Khan, Saeed Ahmad; Cheruth, Abdul Jaleel; Mujib, Abdul; Sharma, Maheshwar Pershad; Srivastava, Prem Shanker

    2011-10-01

    An efficient somatic embryogenesis system has been established in six date palm (Phoenix dactylifera L.) cultivars (Barhee, Zardai, Khalasah, Muzati, Shishi and Zart). Somatic embryogenesis (SE) was growth regulators and cultivars dependent. Friable embryogenic callus was induced from excised shoot tips on MS medium supplemented with various auxins particularly 2,4-dichlorophenoxyacetic acid (2,4-D, 1.5 mg 1(-l)). Suspension culture increased embryogenesis potentiality. Only a-naphthaleneacetic acid (NAA, 0.5 mg 1(-1)) produced somatic embryos in culture. Somatic embryos germinated and converted into plantlets in N(6)-benzyladenine (BAP, 0.75 mg 1(-l)) added medium following a treatment with thidiazuron (TDZ, 1.0 mg 1(-l)) for maturation. Scanning electron microscopy showed early stages of somatic embryo particularly, globular types, and was in masses. Different developing stages of embryogenesis (heart, torpedo and cotyledonary) were observed under histological preparation of embryogenic callus. Biochemical screening at various stages of somatic embryogenesis (embryogenic callus, somatic embryos, matured, germinated embryos and converted plantlets) of date palm cultivars has been conducted and discussed in detail. The result discussed in this paper indicates that somatic embryos were produced in numbers and converted plantlets can be used as a good source of alternative propagation. Genetic modification to the embryo precursor cell may improve the fruit quality and yield further. PMID:23961149

  20. Somatic embryogenesis, scanning electron microscopy, histology and biochemical analysis at different developing stages of embryogenesis in six date palm (Phoenix dactylifera L.) cultivars

    PubMed Central

    Aslam, Junaid; Khan, Saeed Ahmad; Cheruth, Abdul Jaleel; Mujib, Abdul; Sharma, Maheshwar Pershad; Srivastava, Prem Shanker

    2011-01-01

    An efficient somatic embryogenesis system has been established in six date palm (Phoenix dactylifera L.) cultivars (Barhee, Zardai, Khalasah, Muzati, Shishi and Zart). Somatic embryogenesis (SE) was growth regulators and cultivars dependent. Friable embryogenic callus was induced from excised shoot tips on MS medium supplemented with various auxins particularly 2,4-dichlorophenoxyacetic acid (2,4-D, 1.5 mg 1−l). Suspension culture increased embryogenesis potentiality. Only a-naphthaleneacetic acid (NAA, 0.5 mg 1−1) produced somatic embryos in culture. Somatic embryos germinated and converted into plantlets in N6-benzyladenine (BAP, 0.75 mg 1−l) added medium following a treatment with thidiazuron (TDZ, 1.0 mg 1−l) for maturation. Scanning electron microscopy showed early stages of somatic embryo particularly, globular types, and was in masses. Different developing stages of embryogenesis (heart, torpedo and cotyledonary) were observed under histological preparation of embryogenic callus. Biochemical screening at various stages of somatic embryogenesis (embryogenic callus, somatic embryos, matured, germinated embryos and converted plantlets) of date palm cultivars has been conducted and discussed in detail. The result discussed in this paper indicates that somatic embryos were produced in numbers and converted plantlets can be used as a good source of alternative propagation. Genetic modification to the embryo precursor cell may improve the fruit quality and yield further. PMID:23961149

  1. Microarray Analysis of Siberian Ginseng Cyclic Somatic Embryogenesis Culture Systems Provides Insight into Molecular Mechanisms of Embryogenic Cell Cluster Generation

    PubMed Central

    Zhou, Chenguang; Liu, Likun; Li, Chenghao

    2014-01-01

    Four systems of cyclic somatic embryogenesis of Siberian ginseng (Eleutherococcus senticosus Maxim) were used to study the mechanism of embryonic cell cluster generation. The first, direct somatic embryo induction (DSEI), generates secondary embryos directly from the primary somatic embryos; the second, direct embryogenic cell cluster induction (DEC)), induces embryogenic cell clusters directly from somatic embryos in agar medium. Subsequently, we found that when DEC-derived somatic embryos are transferred to suspension culture or a bioreactor culture, only somatic embryos are induced, and embryogenic cell clusters cannot form. Therefore, these new lines were named DEC cultured by liquid medium (ECS) and DEC cultured by bioreactor (ECB), respectively. Transmission electron microscopy showed that DEC epidermal cells contained a variety of inclusions, distinct from other lines. A cDNA library of DEC was constructed, and 1,948 gene clusters were obtained and used as probes. RNA was prepared from somatic embryos from each of the four lines and hybridized to a microarray. In DEC, 7 genes were specifically upregulated compared with the other three lines, and 4 genes were downregulated. EsXTH1 and EsPLT1, which were among the genes upregulated in DEC, were cloned using the rapid amplification of cDNA ends (RACE). Real-time quantitative PCR showed EsXTH1 was more highly expressed in DEC than in other lines throughout the culture cycle, and EsPLT1 expression in DEC increased as culture duration increased, but remained at a low expression level in other lines. These results suggest that EsXTH1 and EsPLT1 may be the essential genes that play important roles during the induction of embryogenic cell clusters. PMID:24743225

  2. The effectiveness of somatic embryogenesis in eliminating the cocoa swollen shoot virus from infected cocoa trees.

    PubMed

    Quainoo, A K; Wetten, A C; Allainguillaume, J

    2008-04-01

    Investigations were undertaken on the use of somatic embryogenesis to generate cocoa swollen shoot virus (CSSV) disease free clonal propagules from infected trees. Polymerase chain reaction (PCR) capillary electrophoresis revealed the presence of CSSV in all the callus tissues induced from the CSSV-infected Amelonado cocoa trees (T1, T2 and T4). The virus was transmitted to primary somatic embryos induced from the infected callus tissues at the rate of 10 (19%), 18 (14%) and 16 (15%) for T1, T2 and T4, respectively. Virus free primary somatic embryos from the infected callus tissues converted into plantlets tested CSSV negative by PCR/capillary electrophoresis 2 years after weaning. Secondary somatic embryos induced from the CSSV-infected primary somatic embryos revealed the presence of viral fragments at the rate of 4 (4%) and 9 (9%) for T2 and T4, respectively. Real-time PCR revealed 23 of the 24 secondary somatic embryos contained no detectable virus. Based on these findings, it is proposed that progressive elimination of the CSSV in infected cocoa trees occurred from primary embryogenesis to secondary embryogenesis. PMID:18294704

  3. Regeneration of Solanum nigrum by Somatic Embryogenesis, Involving Frog Egg-Like Body, a Novel Structure

    PubMed Central

    Xu, Kedong; Chang, Yunxia; Liu, Kun; Wang, Feige; Liu, Zhongyuan; Zhang, Ting; Li, Tong; Zhang, Yi; Zhang, Fuli; Zhang, Ju; Wang, Yan; Niu, Wei; Jia, Shuzhao; Xie, Hengchang; Tan, Guangxuan; Li, Chengwei

    2014-01-01

    A new protocol was established for the regeneration of Solanum nigrum by frog egg-like bodies (FELBs), which are novel somatic embryogenesis (SE) structures induced from the root, stem, and leaf explants. The root, stem, and leaf explants (93.33%, 85.10%, and 100.00%, respectively) were induced to form special embryonic calli on Murashige and Skoog (MS) medium containing 1.0 mg/L 2,4-dichlorophenoxyacetic acid, under dark condition. Further, special embryonic calli from the root, stem, and leaf explants (86.97%, 83.30%, and 99.47%, respectively) were developed into FELBs. Plantlets of FELBs from the three explants were induced in vitro on MS medium supplemented with 5.0 mg/L 6-benzylaminopurine and 0.1 mg/L gibberellic acid, and 100.00% plantlet induction rates were noted. However, plantlet induction in vivo on MS medium supplemented with 20 mg/L thidiazuron showed rates of 38.63%, 15.63%, and 61.30% for the root, stem, and leaf explants, respectively, which were lower than those of the in vitro culture. Morphological and histological analyses of FELBs at different development stages revealed that they are a novel type of SE structure that developed from the mesophyll (leaf) or cortex (stem and root) cells of S. nigrum. PMID:24896090

  4. Control of somatic embryogenesis and embryo development by AP2 transcription factors

    PubMed Central

    El Ouakfaoui, Souad; Schnell, Jaimie; Abdeen, Ashraf; Colville, Adam; Labbé, Hélène; Han, Shuyou; Baum, Bernard; Laberge, Serge

    2010-01-01

    Members of the AP2 family of transcription factors, such as BABY BOOM (BBM), play important roles in cell proliferation and embryogenesis in Arabidopsis thaliana (AtBBM) and Brassica napus (BnBBM) but how this occurs is not understood. We have isolated three AP2 genes (GmBBM1, GmAIL5, GmPLT2) from somatic embryo cultures of soybean, Glycine max (L.) Merr, and discovered GmBBM1 to be homologous to AtBBM and BnBBM. GmAIL5 and GmPLT2 were homologous to Arabidopsis AINTEGUMENTA-like5 (AIL5) and PLETHORA2 (PLT2), respectively. Constitutive expression of GmBBM1 in Arabidopsis induced somatic embryos on vegetative organs and other pleiotropic effects on post-germinative vegetative organ development. Sequence comparisons of BBM orthologues revealed the presence of ten sequence motifs outside of the AP2 DNA-binding domains. One of the motifs, bbm-1, was specific to the BBM-like genes. Deletion and domain swap analyses revealed that bbm-1 was important for somatic embryogenesis and acted cooperatively with at least one other motif, euANT2, in the regulation of somatic embryogenesis and embryo development in transgenic Arabidopsis. The results provide new insights into the mechanisms by which BBM governs embryogenesis. Electronic supplementary material The online version of this article (doi:10.1007/s11103-010-9674-8) contains supplementary material, which is available to authorized users. PMID:20798978

  5. Localization and identification of phenolic compounds in Theobroma cacao L. somatic embryogenesis.

    PubMed

    Alemanno, L; Ramos, T; Gargadenec, A; Andary, C; Ferriere, N

    2003-10-01

    Cocoa breeders and growers continue to face the problem of high heterogeneity between individuals derived from one progeny. Vegetative propagation by somatic embryogenesis could be a way to increase genetic gains in the field. Somatic embryogenesis in cocoa is difficult and this species is considered as recalcitrant. This study was conducted to investigate the phenolic composition of cocoa flowers (the explants used to achieve somatic embryogenesis) and how it changes during the process, by means of histochemistry and conventional chemical techniques. In flowers, all parts contained polyphenolics but their locations were specific to the organ considered. After placing floral explants in vitro, the polyphenolic content was qualitatively modified and maintained in the calli throughout the culture process. Among the new polyphenolics, the three most abundant were isolated and characterized by 1H- and 13C-NMR. They were hydroxycinnamic acid amides: N-trans-caffeoyl-l-DOPA or clovamide, N-trans-p-coumaroyl-l-tyrosine or deoxiclovamide, and N-trans-caffeoyl-l-tyrosine. The same compounds were found also in fresh, unfermented cocoa beans. The synthesis kinetics for these compounds in calli, under different somatic embryogenesis conditions, revealed a higher concentration under non-embryogenic conditions. Given the antioxidant nature of these compounds, they could reflect the stress status of the tissues. PMID:12933367

  6. Somatic embryogenesis and organogenesis from cryopreserved shoot tips of Lilium Oriental hybrid ‘Siberia’

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Somatic embryogenesis and organogenesis were achieved from cryopreserved shoot tips of Lilium Oriental hybrid ‘Siberia’. Shoot tips (1.5-2 mm) were excised from adventitious shoots that were regenerated from basal leaf segments. Precultured shoot tips were then treated with MS containing 0.4 M sucro...

  7. AtLTP1 luciferase expression during carrot somatic embryogenesis.

    PubMed

    Toonen, M A; Verhees, J A; Schmidt, E D; van Kammen, A; de Vries, S C

    1997-11-01

    The carrot (Daucus carota L.) EP2 gene encodes a Lipid Transfer Protein (LTP) which is expressed during protoderm formation in developing embryos. To develop a vital reporter system for gene expression during somatic embryo development a 1.1 kB fragment of the Arabidopsis thaliana LTP1 promoter was fused to the firefly luciferase (LUC) coding sequence. The AtLTP1 luciferase expression pattern in transformed carrot suspension cultures was identical to the expression pattern of the endogenous carrot EP2 gene. Cell tracking experiments revealed that all somatic embryos were derived from AtLTP1 luciferase expressing cell clusters. However, not all cell clusters that expressed the AtLTP1 luciferase reporter gene developed into a somatic embryo, suggesting that initiation of an embryogenic pathway in tissue culture does not always lead to development of a somatic embryo. PMID:9418058

  8. Somatic embryogenesis and enhanced shoot organogenesis in Metabriggsia ovalifolia W. T. Wang

    PubMed Central

    Ouyang, Yao; Chen, Yulu; Lü, Jinfeng; Teixeira da Silva, Jaime A.; Zhang, Xinhua; Ma, Guohua

    2016-01-01

    An efficient protocol providing a dual regeneration pathway via direct shoot organogenesis and somatic embryogenesis for an endangered species, Metabriggsia ovalifolia W. T. Wang, was established from leaf explants. When applied at 2.5 μM, the cytokinins 6-benzyladenine (BA) or thidiazuron (TDZ) and the auxins indole-3-butyric acid (IBA), α-naphthaleneacetic acid (NAA) and indole-3-acetic acid (IAA) could induce shoots when on basal Murashige and Skoog (MS) medium. BA and TDZ could induce more adventitious shoots (19.1 and 31.2/explant, respectively) than NAA (4.6/explant), IBA (5.7/explant) or IAA (6.4/explant). BA and TDZ at 5–10 μM could induce both shoots and somatic embryos. A higher concentration of TDZ (25 μM) induced only somatic embryos (39.8/explant). The same concentration of BA induced both adventitious shoots (23.6/explant) and somatic embryos (9.7/explant). Thus, somatic embryogenesis in this plant needs a high cytokinin concentration (BA; TDZ), as evidenced by histology. Somatic embryos germinated easily when left on the same media, but formed adventitious roots in two weeks on MS supplemented with 0.5 μM NAA, 0.5 μM IBA and 0.1% activated charcoal. Over 93% of plantlets survived following acclimatization and transfer to a mixture of sand and vermiculite (1:1, v/v) in trays. PMID:27090564

  9. Somatic embryogenesis and enhanced shoot organogenesis in Metabriggsia ovalifolia W. T. Wang.

    PubMed

    Ouyang, Yao; Chen, Yulu; Lü, Jinfeng; Teixeira da Silva, Jaime A; Zhang, Xinhua; Ma, Guohua

    2016-01-01

    An efficient protocol providing a dual regeneration pathway via direct shoot organogenesis and somatic embryogenesis for an endangered species, Metabriggsia ovalifolia W. T. Wang, was established from leaf explants. When applied at 2.5 μM, the cytokinins 6-benzyladenine (BA) or thidiazuron (TDZ) and the auxins indole-3-butyric acid (IBA), α-naphthaleneacetic acid (NAA) and indole-3-acetic acid (IAA) could induce shoots when on basal Murashige and Skoog (MS) medium. BA and TDZ could induce more adventitious shoots (19.1 and 31.2/explant, respectively) than NAA (4.6/explant), IBA (5.7/explant) or IAA (6.4/explant). BA and TDZ at 5-10 μM could induce both shoots and somatic embryos. A higher concentration of TDZ (25 μM) induced only somatic embryos (39.8/explant). The same concentration of BA induced both adventitious shoots (23.6/explant) and somatic embryos (9.7/explant). Thus, somatic embryogenesis in this plant needs a high cytokinin concentration (BA; TDZ), as evidenced by histology. Somatic embryos germinated easily when left on the same media, but formed adventitious roots in two weeks on MS supplemented with 0.5 μM NAA, 0.5 μM IBA and 0.1% activated charcoal. Over 93% of plantlets survived following acclimatization and transfer to a mixture of sand and vermiculite (1:1, v/v) in trays. PMID:27090564

  10. [Expression of the WOX and PIN Genes in the Course of Somatic and Zygotic Embryogenesis of a Medicago truncatula].

    PubMed

    Tvorogova, V E; Lebedeva, M A; Lutova, L A

    2015-12-01

    Comparative analysis of the expression of the WOXand PINgenes in ovules and in the course of somatic embryogenesis of Medicago truncatula Gaertn was performed. It was shown that some WOX and PIN genes that had an increased expression level in ovules (MtWOX11-like, STENOFOLIA, MTR_2g015000, and MtPIN10) were also activated in the course of somatic embryogenesis. At the same time, the WOX and PIN genes, the expression level of which was low in ovules, did not show transcription activation associated with somatic embryogenesis. These data allow the assumption that the same regulatory mechanisms can be involved in the control of the early stages of somatic and zygotic embryogenesis. PMID:27055297

  11. Somatic embryogenesis in Arabidopsis thaliana is facilitated by mutations in genes repressing meristematic cell divisions.

    PubMed Central

    Mordhorst, A P; Voerman, K J; Hartog, M V; Meijer, E A; van Went, J; Koornneef, M; de Vries, S C

    1998-01-01

    Embryogenesis in plants can commence from cells other than the fertilized egg cell. Embryogenesis initiated from somatic cells in vitro is an attractive system for studying early embryonic stages when they are accessible to experimental manipulation. Somatic embryogenesis in Arabidopsis offers the additional advantage that many zygotic embryo mutants can be studied under in vitro conditions. Two systems are available. The first employs immature zygotic embryos as starting material, yielding continuously growing embryogenic cultures in liquid medium. This is possible in at least 11 ecotypes. A second, more efficient and reproducible system, employing the primordia timing mutant (pt allelic to hpt, cop2, and amp1), was established. A significant advantage of the pt mutant is that intact seeds, germinated in 2,4-dichlorophenoxyacetic acid (2, 4-D) containing liquid medium, give rise to stable embryonic cell cultures, circumventing tedious hand dissection of immature zygotic embryos. pt zygotic embryos are first distinguishable from wild type at early heart stage by a broader embryonic shoot apical meristem (SAM). In culture, embryogenic clusters originate from the enlarged SAMs. pt somatic embryos had all characteristic embryo pattern elements seen in zygotic embryos, but with higher and more variable numbers of cells. Embryogenic cell cultures were also established from seedling, of other mutants with enlarged SAMs, such as clavata (clv). pt clv double mutants showed additive effects on SAM size and an even higher frequency of seedlings producing embryogenic cell lines. pt clv double mutant plants had very short fasciated inflorescence stems and additive effects on the number of rosette leaves. This suggests that the PT and CLV genes act in independent pathways that control SAM size. An increased population of noncommitted SAM cells may be responsible for facilitated establishment of somatic embryogenesis in Arabidopsis. PMID:9611173

  12. Somatic embryogenesis in immature cotyledons of Manchurian ash (Fraxinus mandshurica Rupr.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Somatic embryogenesis was obtained from immature cotyledon explants that were cultured on half-strength Murashige and Skoog (MS) salts and vitamins with 5.4 uM naphthaleneacetic acid (NAA) and 0.2 uM thidiazuron (TDZ) plus a 4x4 factorial combination of 0,9.8, 34.6, or 49.2 uM indole-3-butyric acid ...

  13. Developmental pathway of somatic embryogenesis in Picea abies as revealed by time-lapse tracking.

    PubMed

    Filonova, L H; Bozhkov, P V; von Arnold, S

    2000-02-01

    Several coniferous species can be propagated via somatic embryogenesis. This is a useful method for clonal propagation, but it can also be used for studying how embryo development is regulated in conifers. However, in conifers it is not known to what extent somatic and zygotic embryos develop similarly, because there has been little research on the origin and development of somatic embryos. A time-lapse tracking technique has been set up, and the development of more than 2000 single cells and few-celled aggregates isolated from embryogenic suspension cultures of Norway spruce (Picea abies L. Karst.) and embedded in thin layers of agarose has been traced. Experiments have shown that somatic embryos develop from proembryogenic masses which pass through a series of three characteristic stages distinguished by cellular organization and cell number (stages I, II and III) to transdifferentiate to somatic embryos. Microscopic inspection of different types of structures has revealed that proembryogenic masses are characterized by high interclonal variation of shape and cellular constitution. In contrast, somatic embryos are morphologically conservative structures, possessing a distinct protoderm-like cell layer as well as embryonal tube cells and suspensor. The lack of staining of the arabinogalactan protein epitope recognized by the monoclonal antibody JIM13 was shown to be an efficient marker for distinguishing proembryogenic masses from somatic embryos. The vast majority of cells in proembryogenic masses expressed this epitope and none of cells in the early somatic embryos. The conditions that promote cell proliferation (i.e. the presence of exogenous auxin and cytokinin), inhibit somatic embryo formation; instead, continuous multiplication of stage I proembryogenic masses by unequal division of embryogenic cells with dense cytoplasm is the prevailing process. Once somatic embryos have formed, their further development to mature forms requires abscisic acid and shares a common histodifferentiation pattern with zygotic embryos. Although the earliest stages of somatic embryo development comparable to proembryogeny could not be characterized, the subsequent developmental processes correspond closely to what occurs in the course of early and late zygotic embryogeny. A model for somatic embryogenesis pathways in Picea abies is presented. PMID:10938831

  14. Stress induced acquisition of somatic embryogenesis in common bean Phaseolus vulgaris L.

    PubMed

    Cabrera-Ponce, José Luis; López, Liliana; León-Ramírez, Claudia G; Jofre-Garfias, Alba E; Verver-y-Vargas, Aurora

    2015-03-01

    Common bean Phaseolus vulgaris L. has been shown to be a recalcitrant plant to induce somatic embryogenesis (SE) under in vitro conditions. We used an alternative strategy to induce SE in common bean based upon the use of a cytokinin (BAP) coupled with osmotic stress adaptation instead of SE response that is induced by auxins. Explants derived from zygotic embryos of common bean were subjected to osmotic stress (sucrose 12 % w/v, 0.5 M) in the presence of BAP 10 mg/L and adenine free base 40 mg/L to induce somatic embryos from specific competent cells of the apical meristem and cotyledonary node. Somatic embryos were obtained from the competent cells in a direct response (direct SE). In a secondary response (secondary SE), those somatic embryos formed proembryogenic masses (PEM) that originated/developed into secondary somatic embryos and showed the SE ontogeny. Maturation of somatic embryos was achieved by using different osmolality media and converted to plants. Full-visible light spectrum was necessary to achieve efficient plant regeneration. Long-term recurrent SE was demonstrated by propagation of PEM at early stages of SE. This protocol is currently being applied for stable genetic transformation by means of Agrobacterium tumefaciens and bioballistics as well as for basic biochemical and molecular biology experiments. PMID:25252886

  15. Somatic embryogenesis and ginsenoside production of Panax ginseng in phytohormone-free medium.

    PubMed

    Shu, W; Yoshimatsu, K; Yamaguchi, H; Shimomura, K

    1999-01-01

    Embryogenic cultures of Panax ginseng were established without using phytohormones. Somatic embryos developed from the roots of an in vitro seedling and from excised leaf and petiole segments cultured in half-macro-salt strength Murashige and Skoog medium. Excised leaf and petiole segments were obtained from in vitro germinated seedlings. Plantlets were subsequently obtained from developing somatic embryos in phytohormone-free media. Shoot formation from somatic embryos was influenced by light intensity. The rate of growth and frequency of embryogenesis were improved when cut-up embryogenic tissues were inoculated into liquid media in the dark. The ginsenoside contents of a 4 year-old field-cultivated root, seedlings from zygotic embryos, somatic embryos and embryogenic tissues were determined and compared. Somatic embryos contained 1.7 times the amount of ginsenoside Rb1 and 2.3 times the amount of ginsenoside Re compared to seedlings from zygotic embryos. Ginsenoside Rd, which was absent in the seedlings derived from zygotic embryos, was detected in somatic embryos. Higher ginsenosides Rd and Rg1 levels were found in embryogenic tissues grown on solid media than in tissues grown in liquid media. The total ginsenoside yields, including the ginsenosides Rb1 and Rg1 levels, of cut-up embryogenic tissues, were higher than those of clump tissues. PMID:10859948

  16. Arabinogalactan proteins and the extracellular matrix surface network during peach palm somatic embryogenesis.

    PubMed

    Steinmacher, Douglas A; Saare-Surminski, Katja; Lieberei, Reinhard

    2012-11-01

    Somatic embryogenesis has been described in peach palm as a reliable method for its in vitro multiplication and conservation. In this study, we evaluated the possible role of arabinogalactan proteins (AGPs) during this morphogenetic pathway. The presence of Yariv reagent, a synthesized chemical antibody that specifically binds AGP molecules, affected somatic embryos and callus development rate, but no effect was observed on fresh weight increment. This substance also had profound effects on embryo morphology: somatic embryos presented loose cells in the protoderm and no signs of polarization could be observed. To better evaluate the role of AGPs, analyses of specific monoclonal antibodies (MAbs) against different AGP epitopes revealed a specific pattern of distribution for each epitope. MAb JIM13 had differential expression and showed intense signal on the embryogenic sector and some immediately adjacent layers. MAb JIM7 against pectin recognized cell walls and a specific layer over the developing somatic embryo, as well as over the shoot meristem region of mature somatic embryos. This corresponds to an extracellular matrix surface network (ECMSN) associated with the development of somatic embryos and closely related to the expression of MAb JIM13. Scanning electron microscopy confirmed the presence of an ECMSN covering a specific group of cells and ultra-structural analyses revealed that the ECMSN had lipophilic substances. PMID:22574975

  17. Conifer somatic embryogenesis: improvements by supplementation of medium with oxidation-reduction agents.

    PubMed

    Pullman, Gerald S; Zeng, Xiaoyan; Copeland-Kamp, Brandi; Crockett, Jonathan; Lucrezi, Jacob; May, Sheldon W; Bucalo, Kylie

    2015-02-01

    A major barrier to the commercialization of somatic embryogenesis technology in loblolly pine (Pinus taeda L.) is recalcitrance of some high-value crosses to initiate embryogenic tissue (ET) and continue early-stage somatic embryo growth. Developing initiation and multiplication media that resemble the seed environment has been shown to decrease this recalcitrance. Glutathione (GSH), glutathione disulfide (GSSG), ascorbic acid and dehydroascorbate analyses were performed weekly throughout the sequence of seed development for female gametophyte and zygotic embryo tissues to determine physiological concentrations. Major differences in stage-specific oxidation-reduction (redox) agents were observed. A simple bioassay was used to evaluate potential growth-promotion of natural and inorganic redox agents added to early-stage somatic embryo growth medium. Compounds showing statistically significant increases in early-stage embryo growth were then tested for the ability to increase initiation of loblolly pine. Low-cost reducing agents sodium dithionite and sodium thiosulfate increased ET initiation for loblolly pine and Douglas fir (Mirb) Franco. Germination medium supplementation with GSSG increased somatic embryo germination. Early-stage somatic embryos grown on medium with or without sodium thiosulfate did not differ in GSH or GSSG content, suggesting that sodium thiosulfate-mediated growth stimulation does not involve GSH or GSSG. We have developed information demonstrating that alteration of the redox environment in vitro can improve ET initiation, early-stage embryo development and somatic embryo germination in loblolly pine. PMID:25716878

  18. Molecular cloning, structural and expression profiling of DlRan genes during somatic embryogenesis in Dimocarpus longan Lour.

    PubMed

    Fang, Zhizhen; Lai, Chengchun; Zhang, Yaling; Lai, Zhongxiong

    2016-01-01

    To clone and examine expression profiles of DlRan genes during somatic embryogenesis in Dimocarpus longan Lour. Thirty cDNA sequences and two genomic sequences encoding DlRan proteins were isolated from longan embryogenic cultures. Structural analysis of DlRan genes revealed that the longan Ran gene family is more expanded than that of Arabidopsis. Expression analysis of DlRan genes during somatic embryogenesis uncovered a high abundance of DlRan genes in early embryogenic cultures and heart- and torpedo-shaped embryos. The expression of DlRan genes in embryogenic calli was affected by exogenous 2,4-dichlorophenoxyacetic acid treatment. DlRan is involved in 2,4-D induced somatic embryogenesis and development of somatic embryos in longan. PMID:27026877

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

    PubMed Central

    2010-01-01

    Background Clonal 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. Results The 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. Conclusions The 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. PMID:20426818

  20. Plant regeneration and somatic embryogenesis from immature embryos derived through interspecific hybridization among different Carica species.

    PubMed

    Azad, Md Abul Kalam; Rabbani, Md Golam; Amin, Latifah

    2012-01-01

    Plant regeneration and somatic embryogenesis through interspecific hybridization among different Carica species were studied for the development of a papaya ringspot virus-resistant variety. The maximum fruit sets were recorded from the cross of the native variety C. papaya cv. Shahi with the wild species C. cauliflora. The highest hybrid embryos were recorded at 90 days after pollination and the embryos were aborted at 150 days after pollination. The immature hybrid embryos were used for plant regeneration and somatic embryogenesis. The 90-day-old hybrid embryos from the cross of C. papaya cv. Shahi × C. cauliflora showed the highest percentage of germination, as well as plant regeneration on growth regulators free culture medium after 7 days pre-incubation on half-strength MS medium supplemented with 0.2 mg/L BAP, 0.5 mg/L NAA and 60 g/L sucrose. The 90-day-old hybrid embryos from the cross of C. papaya cv. Shahi × C. cauliflora produced maximum callus, as well as somatic embryos when cultured on half-strength MS medium containing 5 mg/L 2,4-D, 100 mg/L glutamine, 100 mg/L casein hydrolysate and 60 g/L sucrose. The somatic embryos were transferred into half-strength MS medium containing 0.5 mg/L BAP and 0.2 mg/L NAA and 60 g/L sucrose for maturation. The highest number of regenerated plants per hybrid embryo (10.33) was recorded from the cross of C. papaya cv. Shahi × C. cauliflora. Isoenzyme and dendrogram cluster analysis using UPGMA of the regenerated F(1) plantlets confirmed the presence of the hybrid plantlets. PMID:23235330

  1. Plant Regeneration and Somatic Embryogenesis from Immature Embryos Derived through Interspecific Hybridization among Different Carica Species

    PubMed Central

    Azad, Md. Abul Kalam; Rabbani, Md. Golam; Amin, Latifah

    2012-01-01

    Plant regeneration and somatic embryogenesis through interspecific hybridization among different Carica species were studied for the development of a papaya ringspot virus-resistant variety. The maximum fruit sets were recorded from the cross of the native variety C. papaya cv. Shahi with the wild species C. cauliflora. The highest hybrid embryos were recorded at 90 days after pollination and the embryos were aborted at 150 days after pollination. The immature hybrid embryos were used for plant regeneration and somatic embryogenesis. The 90-day-old hybrid embryos from the cross of C. papaya cv. Shahi × C. cauliflora showed the highest percentage of germination, as well as plant regeneration on growth regulators free culture medium after 7 days pre-incubation on half-strength MS medium supplemented with 0.2 mg/L BAP, 0.5 mg/L NAA and 60 g/L sucrose. The 90-day-old hybrid embryos from the cross of C. papaya cv. Shahi × C. cauliflora produced maximum callus, as well as somatic embryos when cultured on half-strength MS medium containing 5 mg/L 2,4-D, 100 mg/L glutamine, 100 mg/L casein hydrolysate and 60 g/L sucrose. The somatic embryos were transferred into half-strength MS medium containing 0.5 mg/L BAP and 0.2 mg/L NAA and 60 g/L sucrose for maturation. The highest number of regenerated plants per hybrid embryo (10.33) was recorded from the cross of C. papaya cv. Shahi × C. cauliflora. Isoenzyme and dendrogram cluster analysis using UPGMA of the regenerated F1 plantlets confirmed the presence of the hybrid plantlets. PMID:23235330

  2. Influence of low temperature preincubation on somatic embryogenesis and ethylene emanation from orchardgrass leaves

    NASA Technical Reports Server (NTRS)

    Tomaszewski, Z. Jr; Kuklin, A. I.; Sams, C. E.; Conger, B. V.

    1994-01-01

    The objectives of this study were to determine the effects of low temperature (4 degrees C) preincubation on somatic embryogenesis from orchardgrass (Dactylis glomerata L.) leaf cultures and to relate these effects to ethylene emanation during the preincubation and incubation periods. Experiments were also conducted with an ethylene biosynthesis inhibitor aminooxyacetic acid (AOA). Segments from the innermost two leaves were cultured on SH medium with 30 micromoles dicamba at 4 degrees C for 1 to 7 d before transfer to 21 degrees C. Results from a paired design showed that the embryogenic response of leaf segments preincubated at 4 degrees C was equal or superior to nonpreincubated leaves at all time periods. Ethylene emanation was decreased during the low temperature incubation. Transfer of leaf segments from 4 degrees C to 21 degrees C was accompanied by a burst of ethylene which rose to control levels within 30 min. AOA at 20 and 40 micromoles decreased ethylene emanation but did not stimulate the embryogenic response. We conclude that the stimulation of somatic embryogenesis by low temperature is probably due to factors other than suppression of ethylene biosynthesis.

  3. Histocytological Analysis of Callogenesis and Somatic Embryogenesis from Cell Suspensions of Date Palm (Phoenix dactylifera)

    PubMed Central

    SANÉ, D.; ABERLENC-BERTOSSI, F.; GASSAMA-DIA, Y. K.; SAGNA, M.; TROUSLOT, M. F.; DUVAL, Y.; BORGEL, A.

    2006-01-01

    • Background and Aims The date palm is a dioecious perennial species of the Arecaceae for which in vitro micropropagation is essential to ensure the renewal of palm plantations. This study presents a histocytological analysis of the traditional Mauritanian Amsekhsi cultivar beginning from the initial callogenesis and continuing up to the establishment of the cellular embryogenic cell suspensions. The formation of somatic embryos and their development into rooted plants are also described. • Methods Foliar segments of seedlings cultured in the presence of 2,4-D produced primary calli that were chopped to produce fine friable granular calli that subsequently produced cellular suspensions when transferred to liquid medium. The somatic proembryos that developed after removal of the 2,4-D were plated on agar medium where they developed into rooted plants. Thin sections of tissue fragments taken at each stage of the process were stained using Periodic Acid Schiff and Naphthol Blue-Black. • Key Results The first cellular divisions were localized close to the vascular vessels of the leaf. The primary calli were obtained within 2 months. Fine friable granular calli grew quickly after the primary calli were chopped. Individual embryogenic cells were identified that rapidly started to divide and developed into globular proembryos. In addition, in the microcalli, breaking zones appeared in the thick pectocellulosic walls which delimited the pluricellular proembryos. The anatomy of somatic embryos is similar to that of zygotic embryos despite a deficit in the accumulation of intracellular proteins. When rooted with NAA, the vitroplants developed a strong orthotropic taproot. • Conclusions This study contributes to understanding the whole process of somatic embryogenesis, but two specific questions remain to be answered: what factors are involved in the reactivation of the somatic cells at the beginning of the initial callogenesis, and why do the somatic embryos not accumulate proteins in their tissues during maturation? PMID:16740588

  4. The Agrobacterium rhizogenes rolC-gene-induced somatic embryogenesis and shoot organogenesis in Panax ginseng transformed calluses.

    PubMed

    Gorpenchenko, T Y; Kiselev, K V; Bulgakov, V P; Tchernoded, G K; Bragina, E A; Khodakovskaya, M V; Koren, O G; Batygina, T B; Zhuravlev, Yu N

    2006-02-01

    Expression of the Agrobacterium rhizogenes rolC gene in Panax ginseng callus cells results in formation of tumors that are capable to form roots. The selection of non-root forming tumor clusters yielded the embryogenic 2c3 callus line, which formed somatic embryos and shoots independently of external growth factors. Although the 2c3 somatic embryos developed through a typical embryogenesis process, they terminated prematurely and repeatedly formed adventitious shoot meristems and embryo-like structures. A part of the shoots and somatic embryos formed enlarged and fasciated meristems. This is the first indication of the rolC gene embryogenic effect and, to our knowledge, the first indication that a single gene of non-plant origin can induce somatic embryogenesis in plants. PMID:16136334

  5. Somatic embryogenesis and plant regeneration from immature zygotic embryos of Hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.).

    PubMed

    Taniguchi, T; Kurita, M; Itahana, N; Kondo, T

    2004-08-01

    We established a plant regeneration system for Hinoki cypress (Chamaecyparis obtusa) via somatic embryogenesis. Embryogenic tissues were successfully induced on three kinds of Smith media from megagametophyte explants containing pre-cotyledonary embryos of C. obtusa plus-trees. Factors affecting somatic embryo maturation were examined. The concentration of polyethylene glycol 4000 in the medium was a critical factor for embryo maturation and its effective concentration was 150 g/l. The addition of 30 g/l maltose to the medium had a positive effect on embryo maturation, but sucrose was ineffective. The mature somatic embryos germinated at a germination frequency of approximately 60%, and the presence of activated charcoal was effective in stimulating plantlet growth. The plantlets acclimatized successfully in a greenhouse. To our knowledge, this is first report describing details of a plant regeneration method for C. obtusa via somatic embryogenesis. PMID:15141322

  6. First Report of Plant Regeneration via Somatic Embryogenesis from Shoot Apex-derived Callus of Hedychium muluense

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plants were successfully regenerated via somatic embryogenesis from shoot apex-derived callus of Hedychium muluense R.M. Smith, an important monocotyledonous ornamental ginger plant. Callus was induced on a modified Murashige and Skoog (MS) medium supplemented with 9.05 µM 2-4, D and 4.6µM kinetin. ...

  7. Differential gene expression of cotton cultivar CCRI24 during somatic embryogenesis.

    PubMed

    Wu, Xiuming; Li, Fuguang; Zhang, Chaojun; Liu, Chuanliang; Zhang, Xueyan

    2009-08-15

    Regeneration is an essential step for recovery of transgenic plants following gene transfer. However, most cotton cultivars fail to respond to the current regeneration protocols for cotton. This hinders the use of gene transfer technology to improve this crop. Identification of the genes involved in cotton somatic embryogenesis (SE) may provide information that will help to improve regeneration protocols. To investigate the genes expressed during cotton SE, we constructed a suppression subtractive hybridization (SSH) library using cDNA from nonembryogenic callus as driver and those from embryogenic callus as tester. From this library, 368 cDNA clones that hybridized conspicuously to the forward-subtracted and unsubtracted tester probes, but not to the reverse-subtracted or unsubtracted driver probes, were obtained and sequenced. Among the 241 putative unigenes, the functions of 152 genes (63%) could be assigned using existing databases. In addition to many previously reported SE-related genes, some new genes, such as members of ethylene pathway and auxin pathway, were discovered in our library. The expression of eight genes, including an Aux/IAA responsive gene, was analyzed by reverse-transcription-polymerase chain reaction and found to be upregulated during the SE. This is in agreement with previous studies showing that embryogenesis involves auxin signaling. PMID:19328593

  8. Transcriptome profiling and digital gene expression by deep sequencing in early somatic embryogenesis of endangered medicinal Eleutherococcus senticosus Maxim.

    PubMed

    Tao, Lei; Zhao, Yue; Wu, Ying; Wang, Qiuyu; Yuan, Hongmei; Zhao, Lijuan; Guo, Wendong; You, Xiangling

    2016-03-01

    Somatic embryogenesis (SE) has been studied as a model system to understand molecular events in physiology, biochemistry, and cytology during plant embryo development. In particular, it is exceedingly difficult to access the morphological and early regulatory events in zygotic embryos. To understand the molecular mechanisms regulating early SE in Eleutherococcus senticosus Maxim., we used high-throughput RNA-Seq technology to investigate its transcriptome. We obtained 58,327,688 reads, which were assembled into 75,803 unique unigenes. To better understand their functions, the unigenes were annotated using the Clusters of Orthologous Groups, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes databases. Digital gene expression libraries revealed differences in gene expression profiles at different developmental stages (embryogenic callus, yellow embryogenic callus, global embryo). We obtained a sequencing depth of >5.6 million tags per sample and identified many differentially expressed genes at various stages of SE. The initiation of SE affected gene expression in many KEGG pathways, but predominantly that in metabolic pathways, biosynthesis of secondary metabolites, and plant hormone signal transduction. This information on the changes in the multiple pathways related to SE induction in E. senticosus Maxim. embryogenic tissue will contribute to a more comprehensive understanding of the mechanisms involved in early SE. Additionally, the differentially expressed genes may act as molecular markers and could play very important roles in the early stage of SE. The results are a comprehensive molecular biology resource for investigating SE of E. senticosus Maxim. PMID:26657036

  9. Analysis of trace elements during different developmental stages of somatic embryogenesis in Plantago ovata Forssk using energy dispersive X-ray fluorescence.

    PubMed

    Saha, Priyanka; Raychaudhuri, Sarmistha Sen; Sudarshan, Mathummal; Chakraborty, Anindita

    2010-06-01

    Energy dispersive X-ray fluorescence (ED-XRF) technique has been used for the determination of trace element profile during different developmental stages of somatic embryogenic callus of an economically important medicinal plant, Plantago ovata Forssk. Somatic embryogenesis is a plant tissue culture-based technique, which is used for plant regeneration and crop improvement. In the present investigation, elemental content was analysed using ED-XRF technique during different developmental stages and also determine the effect of additives--casein hydrolysate and coconut water on the trace elemental profile of embryogenic callus tissue of P. ovata. Subsequent experiments showed significant alteration in the concentration of K, Ca, Mn, Fe, Zn, Cu, Br, and Sr in both the embryogenic and non-embryogenic callus. Higher K, Ca, Fe, Cu, and Zn accumulation was in embryogenic tissue stage compared to other stages, suggesting these elements are crucial for successful embryogenesis. The results suggest that this information could be useful for formulating a media for in vitro embryo induction of P. ovata. PMID:19696971

  10. Unusual patterns of somatic embryogenesis in the domesticated carrot: developmental effects of exogenous auxins and auxin transport inhibitors.

    PubMed

    Schiavone, F M; Cooke, T J

    1987-06-01

    The effects of various exogenous auxins and polar auxin transport inhibitors on somatic embryogenesis in carrot cultures were investigated. Indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid do not disrupt the sequence or the polarity of individual stages in embryo development, but tend to cause developing embryos to revert to undifferentiated callus, with increasing frequency in later embryo stages. The transport inhibitors, N-(1-naphthyl)phthalamic acid and 2,3,5-triiodobenzoic acid, block morphological transitions to the subsequent stage; for example, they cause the formation of enlarged globular and oblong embryos. Heart embryos in these treatments usually develop additional lateral growth axes. These results shed light on the role of auxin and its polar transport in somatic embryogenesis. PMID:3607884

  11. Glutathione-S-Transferase is Detected During Somatic Embryogenesis in Chicory

    PubMed Central

    Galland, Rachel; Blervacq, Anne-Sophie; Blassiau, Christelle; Smagghe, Benoît; Decottignies, Jean-Pierre

    2007-01-01

    Glutathione S-tranferases (GSTs) are a heterogeneous family of proteins, which perform diverse pivotal catalytic and non-enzymatic functions during plant development and in plant stress responses. Previous studies have shown that a GST activity (EC 2.5.1.18) is closely linked with the precocious phases of somatic embryogenesis in leaf tissues of an interspecific chicory hybrid (Cichorium intybus L. var. sativa × C. endivia L. var. latifolia). In order to learn more about the involvement of this enzyme in this process, in situ-hybridization as well as immunolocalization were performed in parallel. GST-mRNAs and proteins were colocalized in small veins, particularly in young protoxylem cell walls. During cell reactivation, the in situ and protein signals became less intense and were associated with chloroplasts. The GST-mRNAs and corresponding proteins were not always colocalized in the same tissues. While high amounts of transcripts could be detected in multicellular embryos, the proteins were not well labeled. Our results indicated that GSTs belong to a complex anti-oxidant mechanism within the cell, and also at the cell wall level. GSTs presence in reactivated cell and multicellular embryos is discussed in relation to redox cell status. PMID:19516999

  12. A Chimeric Arabinogalactan Protein Promotes Somatic Embryogenesis in Cotton Cell Culture1[W][OA

    PubMed Central

    Poon, Simon; Heath, Robyn Louise; Clarke, Adrienne Elizabeth

    2012-01-01

    Arabinogalactan proteins (AGPs) are a family of extracellular plant proteoglycans implicated in many aspects of plant growth and development, including in vitro somatic embryogenesis (SE). We found that specific AGPs were produced by cotton (Gossypium hirsutum) calli undergoing SE and that when these AGPs were isolated and incorporated into tissue culture medium, cotton SE was promoted. When the AGPs were partly or fully deglycosylated, SE-promoting activity was not diminished. Testing of AGPs separated by reverse-phase high-performance liquid chromatography revealed that the SE-promoting activity resided in a hydrophobic fraction. We cloned a full-length complementary DNA (cotton PHYTOCYANIN-LIKE ARABINOGALACTAN-PROTEIN1 [GhPLA1]) that encoded the protein backbone of an AGP in the active fraction. It has a chimeric structure comprising an amino-terminal signal sequence, a phytocyanin-like domain, an AGP-like domain, and a hydrophobic carboxyl-terminal domain. Recombinant production of GhPLA1 in tobacco (Nicotiana tabacum) cells enabled us to purify and analyze a single glycosylated AGP and to demonstrate that this chimeric AGP promotes cotton SE. Furthermore, the nonglycosylated phytocyanin-like domain from GhPLA1, which was bacterially produced, also promoted SE, indicating that the glycosylated AGP domain was unnecessary for in vitro activity. PMID:22858635

  13. Doubled haploid production from Spanish onion (Allium cepa L.) germplasm: embryogenesis induction, plant regeneration and chromosome doubling.

    PubMed

    Fayos, Oreto; Vallés, María P; Garcés-Claver, Ana; Mallor, Cristina; Castillo, Ana M

    2015-01-01

    The use of doubled haploids in onion breeding is limited due to the low gynogenesis efficiency of this species. Gynogenesis capacity from Spanish germplasm, including the sweet cultivar Fuentes de Ebro, the highly pungent landrace BGHZ1354 and the two Valenciana type commercial varieties Recas and Rita, was evaluated and optimized in this study. The OH-1 population, characterized by a high gynogenesis induction, was used as control. Growing conditions of the donor plants were tested with a one-step protocol and field plants produced a slightly higher percentage of embryogenesis induction than growth chamber plants. A one-step protocol was compared with a two-step protocol for embryogenesis induction. Spanish germplasm produced a 2-3 times higher percentage of embryogenesis with the two-step protocol, Recas showing the highest percentage (2.09%) and Fuentes de Ebro the lowest (0.53%). These percentages were significantly lower than those from the OH-1 population, with an average of 15% independently of the protocol used. The effect of different containers on plant regeneration was tested using both protocols. The highest percentage of acclimated plants was obtained with the two-step protocol in combination with Eco2box (70%), whereas the lowest percentage was observed with glass tubes in the two protocols (20-23%). Different amiprofos-methyl (APM) treatments were applied to embryos for chromosome doubling. A similar number of doubled haploid plants were recovered with 25 or 50 μM APM in liquid medium. However, the application of 25 μM in solid medium for 24 h produced the highest number of doubled haploid plants. Somatic regeneration from flower buds of haploid and mixoploid plants proved to be a successful approach for chromosome doubling, since diploid plants were obtained from the four regenerated lines. In this study, doubled haploid plants were produced from the four Spanish cultivars, however further improvements are needed to increase their gynogenesis efficiency. PMID:26074944

  14. Doubled haploid production from Spanish onion (Allium cepa L.) germplasm: embryogenesis induction, plant regeneration and chromosome doubling

    PubMed Central

    Fayos, Oreto; Vallés, María P.; Garcés-Claver, Ana; Mallor, Cristina; Castillo, Ana M.

    2015-01-01

    The use of doubled haploids in onion breeding is limited due to the low gynogenesis efficiency of this species. Gynogenesis capacity from Spanish germplasm, including the sweet cultivar Fuentes de Ebro, the highly pungent landrace BGHZ1354 and the two Valenciana type commercial varieties Recas and Rita, was evaluated and optimized in this study. The OH-1 population, characterized by a high gynogenesis induction, was used as control. Growing conditions of the donor plants were tested with a one-step protocol and field plants produced a slightly higher percentage of embryogenesis induction than growth chamber plants. A one-step protocol was compared with a two-step protocol for embryogenesis induction. Spanish germplasm produced a 2–3 times higher percentage of embryogenesis with the two-step protocol, Recas showing the highest percentage (2.09%) and Fuentes de Ebro the lowest (0.53%). These percentages were significantly lower than those from the OH-1 population, with an average of 15% independently of the protocol used. The effect of different containers on plant regeneration was tested using both protocols. The highest percentage of acclimated plants was obtained with the two-step protocol in combination with Eco2box (70%), whereas the lowest percentage was observed with glass tubes in the two protocols (20–23%). Different amiprofos-methyl (APM) treatments were applied to embryos for chromosome doubling. A similar number of doubled haploid plants were recovered with 25 or 50 μM APM in liquid medium. However, the application of 25 μM in solid medium for 24 h produced the highest number of doubled haploid plants. Somatic regeneration from flower buds of haploid and mixoploid plants proved to be a successful approach for chromosome doubling, since diploid plants were obtained from the four regenerated lines. In this study, doubled haploid plants were produced from the four Spanish cultivars, however further improvements are needed to increase their gynogenesis efficiency. PMID:26074944

  15. Somatic embryogenesis of Echinodorus orisis L. and the kinetic changes of the endogenous hormones contents during the embryogenetic process.

    PubMed

    Xing, D; Zhao, Y; Huang, C

    1999-01-01

    Somatic embryogenesis was achieved in young Leaf cuttings of Echinodorus orisis L., an aquatic ornamental plant, in a short period (25 days). Among the cytokinins and their combinations tested, 6-BA (1 mg/L) and Zt (1 mg/L) in MS medium induced the highest efficiency (100%) of somatic embryogenesis, with a maximum of 4.87 embryoids per explant. Roots instead of somatic embryos were formed when NAA (0.5 mg/L) was added to MS medium containing Zt (1 mg/L). Matured embryoids were germinated and rooted in MS medium with IAA (1 mg/L) after 5 days cultivation. Seventy-two percent of the rooted plantlets transplanted survived in the aquarium. The endogenous hormone contents in various stages of somatic embryogenetic process were measured by HPLC. The concentrations of all the hormones tested were about 2 times that of the cuttings from the untreated fresh leaves after 10 days incubation. Meanwhile, the concentration of IAA presents two peaks after 10 and 25 days of cultivation, respectively. The cytokinin (Zt and ZR) peak, about 8 times more than CK, appeared in 15 days cultivation when the heart-shaped embryos formed. The fluctuation of the GA3 concentration was very similar to that of cytokinin. The ABA, however, remains stable at quite high concentration after 10 days of cultivation. PMID:10668136

  16. Jasmonic acid is a downstream component in the modulation of somatic embryogenesis by Arabidopsis Class 2 phytoglobin

    PubMed Central

    Mira, Mohamed M.; Wally, Owen S. D.; Elhiti, Mohamed; El-Shanshory, Adel; Reddy, Dhadi S.; Hill, Robert D.; Stasolla, Claudio

    2016-01-01

    Previous studies have shown that the beneficial effect of suppression of the Arabidopsis phytoglobin 2 gene, PGB2, on somatic embryogenesis occurs through the accumulation of nitric oxide (NO) within the embryogenic cells originating from the cultured explant. NO activates the expression of Allene oxide synthase (AOS) and Lipoxygenase 2 (LOX2), genes encoding two key enzymes of the jasmonic acid (JA) biosynthetic pathway, elevating JA content within the embryogenic tissue. The number of embryos in the single aos1-1 mutant and pgb2-aos1-1 double mutant declined, and was not rescued by increasing levels of NO stimulating embryogenesis in wild-type tissue. NO also influenced JA responses by up-regulating PLANT DEFENSIN 1 (PDF1) and JASMONATE-ZIM-PROTEIN (JAZ1), as well as down-regulating MYC2. The NO and JA modulation of MYC2 and JAZ1 controlled embryogenesis. Ectopic expression of JAZ1 or suppression of MYC2 promoted the formation of somatic embryos, while repression of JAZ1 and up-regulation of MYC2 reduced the embryogenic performance. Sustained expression of JAZ1 induced the transcription of several indole acetic acid (IAA) biosynthetic genes, resulting in higher IAA levels in the embryogenic cells. Collectively these data fit a model integrating JA in the PGB2 regulation of Arabidopsis embryogenesis. Suppression of PGB2 increases JA through NO. Elevated levels of JA repress MYC2 and induce JAZ1, favoring the accumulation of IAA in the explants and the subsequent production of somatic embryos. PMID:26962208

  17. Rorippa indica Regeneration via Somatic Embryogenesis Involving Frog Egg-like Bodies Efficiently Induced by the Synergy of Salt and Drought Stresses.

    PubMed

    Xu, Kedong; Chang, Yunxia; Zhang, Yi; Liu, Kun; Zhang, Ju; Wang, Wei; Li, Zhanshuai; Wu, Jianxin; Ma, Shuya; Xin, Yuexing; Li, Chunjing; Zhou, Qianbei; Qiu, Hanhan; Pi, Yumei; Wang, Youwei; Tan, Guangxuan; Li, Chengwei

    2016-01-01

    Frog egg-like bodies (FELBs), novel somatic embryogenesis (SE) structures first observed in Solanum nigrum, were induced in Rorippa indica. NaCl-mediated salt and mannitol-mimicked drought stresses induced FELBs in R. indica, which is very different from the induction by plant growth regulators (PGRs) under low light condition that was used in S. nigrum FELB induction. It demonstrated that NaCl or mannitol supplements alone could induce FELBs in R. indica, but with low induction rates, while the synergy of NaCl and mannitol significantly increased the FELB induction rates. For the combination of 5.0 g/L mannitol and 10.0 g/L NaCl the highest FELB induction rate (100%) was achieved. It suggests that the synergy of drought and salt stresses can replace PGRs to induce FELBs in R. indica. On medium supplemented with 1.0 mg/L gibberellic acid all the inoculated in vitro FELBs developed into multiple plantlets. Morphological and histological analyses confirmed the identity of FELBs induced in R. indica and revealed that FELBs originate from root cortex cells. PMID:26796345

  18. Rorippa indica Regeneration via Somatic Embryogenesis Involving Frog Egg-like Bodies Efficiently Induced by the Synergy of Salt and Drought Stresses

    PubMed Central

    Xu, Kedong; Chang, Yunxia; Zhang, Yi; Liu, Kun; Zhang, Ju; Wang, Wei; Li, Zhanshuai; Wu, Jianxin; Ma, Shuya; Xin, Yuexing; Li, Chunjing; Zhou, Qianbei; Qiu, Hanhan; Pi, Yumei; Wang, Youwei; Tan, Guangxuan; Li, Chengwei

    2016-01-01

    Frog egg-like bodies (FELBs), novel somatic embryogenesis (SE) structures first observed in Solanum nigrum, were induced in Rorippa indica. NaCl-mediated salt and mannitol-mimicked drought stresses induced FELBs in R. indica, which is very different from the induction by plant growth regulators (PGRs) under low light condition that was used in S. nigrum FELB induction. It demonstrated that NaCl or mannitol supplements alone could induce FELBs in R. indica, but with low induction rates, while the synergy of NaCl and mannitol significantly increased the FELB induction rates. For the combination of 5.0 g/L mannitol and 10.0 g/L NaCl the highest FELB induction rate (100%) was achieved. It suggests that the synergy of drought and salt stresses can replace PGRs to induce FELBs in R. indica. On medium supplemented with 1.0 mg/L gibberellic acid all the inoculated in vitro FELBs developed into multiple plantlets. Morphological and histological analyses confirmed the identity of FELBs induced in R. indica and revealed that FELBs originate from root cortex cells. PMID:26796345

  19. Genome-wide identification, classification and analysis of HD-ZIP gene family in citrus, and its potential roles in somatic embryogenesis regulation.

    PubMed

    Ge, Xiao-Xia; Liu, Zheng; Wu, Xiao-Meng; Chai, Li-Jun; Guo, Wen-Wu

    2015-12-10

    The homeodomain-leucine zipper (HD-Zip) transcription factors, which belong to a class of Homeobox proteins, has been reported to be involved in different biological processes of plants, including growth and development, photomorphogenesis, flowering, fruit ripening and adaptation responses to environmental stresses. In this study, 27 HD-Zip genes (CsHBs) were identified in Citrus. Based on the phylogenetic analysis and characteristics of individual gene or protein, the HD-Zip gene family in Citrus can be classified into 4 subfamilies, i.e. HD-Zip I, HD-Zip II, HD-Zip III, and HD-Zip IV containing 16, 2, 4, and 5 members respectively. The digital expression patterns of 27 HD-Zip genes were analyzed in the callus, flower, leaf and fruit of Citrus sinensis. The qRT-PCR and RT-PCR analyses of six selected HD-Zip genes were performed in six citrus cultivars with different embryogenic competence and in the embryo induction stages, which revealed that these genes were differentially expressed and might be involved in citrus somatic embryogenesis (SE). The results exhibited that the expression of CsHB1 was up-regulated in somatic embryo induction process, and its expression was higher in citrus cultivars with high embryogenic capacity than in cultivars recalcitrant to form somatic embryos. Moreover, a microsatellite site of three nucleotide repeats was found in CsHB1 gene among eighteen citrus genotypes, indicating the possible association of CsHB1 gene to the capacity of callus induction. PMID:26232336

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

    PubMed

    Shim, Y S; Kasha, K J; Simion, E; Letarte, J

    2006-08-01

    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. PMID:16937058

  1. New Insights into Somatic Embryogenesis: LEAFY COTYLEDON1, BABY BOOM1 and WUSCHEL-RELATED HOMEOBOX4 Are Epigenetically Regulated in Coffea canephora

    PubMed Central

    Nic-Can, Geovanny I.; López-Torres, Adolfo; Barredo-Pool, Felipe; Wrobel, Kazimierz; Loyola-Vargas, Víctor M.; Rojas-Herrera, Rafael; De-la-Peña, Clelia

    2013-01-01

    Plant cells have the capacity to generate a new plant without egg fertilization by a process known as somatic embryogenesis (SE), in which differentiated somatic cells can form somatic embryos able to generate a functional plant. Although there have been advances in understanding the genetic basis of SE, the epigenetic mechanism that regulates this process is still unknown. Here, we show that the embryogenic development of Coffea canephora proceeds through a crosstalk between DNA methylation and histone modifications during the earliest embryogenic stages of SE. We found that low levels of DNA methylation, histone H3 lysine 9 dimethylation (H3K9me2) and H3K27me3 change according to embryo development. Moreover, the expression of LEAFY COTYLEDON1 (LEC1) and BABY BOOM1 (BBM1) are only observed after SE induction, whereas WUSCHEL-RELATED HOMEOBOX4 (WOX4) decreases its expression during embryo maturation. Using a pharmacological approach, it was found that 5-Azacytidine strongly inhibits the embryogenic response by decreasing both DNA methylation and gene expression of LEC1 and BBM1. Therefore, in order to know whether these genes were epigenetically regulated, we used Chromatin Immunoprecipitation (ChIP) assays. It was found that WOX4 is regulated by the repressive mark H3K9me2, while LEC1 and BBM1 are epigenetically regulated by H3K27me3. We conclude that epigenetic regulation plays an important role during somatic embryogenic development, and a molecular mechanism for SE is proposed. PMID:23977240

  2. Maize miRNA and target regulation in response to hormone depletion and light exposure during somatic embryogenesis

    PubMed Central

    Chávez-Hernández, Elva C.; Alejandri-Ramírez, Naholi D.; Juárez-González, Vasti T.; Dinkova, Tzvetanka D.

    2015-01-01

    Maize somatic embryogenesis (SE) is induced from the immature zygotic embryo in darkness and under the appropriate hormones' levels. Small RNA expression is reprogrammed and certain miRNAs become particularly enriched during induction while others, characteristic to the zygotic embryo, decrease. To explore the impact of different environmental cues on miRNA regulation in maize SE, we tested specific miRNA abundance and their target gene expression in response to photoperiod and hormone depletion for two different maize cultivars (VS-535 and H-565). The expression levels of miR156, miR159, miR164, miR168, miR397, miR398, miR408, miR528, and some predicted targets (SBP23, GA-MYB, CUC2, AGO1c, LAC2, SOD9, GR1, SOD1A, PLC) were examined upon staged hormone depletion in the presence of light photoperiod or darkness. Almost all examined miRNA, except miR159, increased upon hormone depletion, regardless photoperiod absence/presence. miR528, miR408, and miR398 changed the most. On the other hand, expression of miRNA target genes was strongly regulated by the photoperiod exposure. Stress-related miRNA targets showed greater differences between cultivars than development-related targets. miRNA/target inverse relationship was more frequently observed in darkness than light. Interestingly, miR528, but not miR159, miR168 or miR398, was located on polyribosome fractions suggesting a role for this miRNA at the level of translation. Overall our results demonstrate that hormone depletion exerts a great influence on specific miRNA expression during plant regeneration independently of light. However, their targets are additionally influenced by the presence of photoperiod. The reproducibility or differences observed for particular miRNA-target regulation between two different highly embryogenic genotypes provide clues for conserved miRNA roles within the SE process. PMID:26257760

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

    PubMed Central

    2011-01-01

    Background Hydroxyproline 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. Results Immunofluorescence 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. Conclusions These 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. PMID:21349190

  4. A Lower pH Value Benefits Regeneration of Trichosanthes kirilowii by Somatic Embryogenesis, Involving Rhizoid Tubers (RTBs), a Novel Structure

    PubMed Central

    Xu, Ke-dong; Chang, Yun-xia; Zhang, Ju; Wang, Pei-long; Wu, Jian-xin; Li, Yan-yan; Wang, Xiao-wen; Wang, Wei; Liu, Kun; Zhang, Yi; Yu, De-shui; Liao, Li-bing; Li, Yi; Ma, Shu-ya; Tan, Guang-xuan; Li, Cheng-wei

    2015-01-01

    A new approach was established for the regeneration of Trichosanthes kirilowii from root, stem, and leaf explants by somatic embryogenesis (SE), involving a previously unreported SE structure, rhizoid tubers (RTBs). During SE, special rhizoids were first induced from root, stem, and leaf explants with average rhizoid numbers of 62.33, 40.17, and 11.53 per explant, respectively, on Murashige and Skoog (MS) medium (pH 4.0) supplemented with 1.0 mg/L 1-naphthaleneacetic acid (NAA) under dark conditions. Further, one RTB was formed from each of the rhizoids on MS medium (pH 4.0) supplemented with 20 mg/L thidiazuron (TDZ) under light conditions. In the suitable range (pH 4.0–9.0), a lower pH value increased the induction of rhizoids and RTBs. Approximately 37.77, 33.47, and 31.07% of in vivo RTBs from root, stem, and leaf explants, respectively, spontaneously developed into multiple plantlets on the same MS medium (supplemented with 20 mg/L TDZ) for induction of RTBs, whereas >95.00% of in vitro RTBs from each kind of explant developed into multiple plantlets on MS medium supplemented with 5.0 mg/L 6-benzylaminopurine (BAP). Morphological and histological analyses revealed that RTB is a novel type of SE structure that develops from the cortex cells of rhizoids. PMID:25744384

  5. A lower pH value benefits regeneration of Trichosanthes kirilowii by somatic embryogenesis, involving rhizoid tubers (RTBs), a novel structure.

    PubMed

    Xu, Ke-dong; Chang, Yun-xia; Zhang, Ju; Wang, Pei-long; Wu, Jian-xin; Li, Yan-yan; Wang, Xiao-wen; Wang, Wei; Liu, Kun; Zhang, Yi; Yu, De-shui; Liao, Li-bing; Li, Yi; Ma, Shu-ya; Tan, Guang-xuan; Li, Cheng-wei

    2015-01-01

    A new approach was established for the regeneration of Trichosanthes kirilowii from root, stem, and leaf explants by somatic embryogenesis (SE), involving a previously unreported SE structure, rhizoid tubers (RTBs). During SE, special rhizoids were first induced from root, stem, and leaf explants with average rhizoid numbers of 62.33, 40.17, and 11.53 per explant, respectively, on Murashige and Skoog (MS) medium (pH 4.0) supplemented with 1.0 mg/L 1-naphthaleneacetic acid (NAA) under dark conditions. Further, one RTB was formed from each of the rhizoids on MS medium (pH 4.0) supplemented with 20 mg/L thidiazuron (TDZ) under light conditions. In the suitable range (pH 4.0-9.0), a lower pH value increased the induction of rhizoids and RTBs. Approximately 37.77, 33.47, and 31.07% of in vivo RTBs from root, stem, and leaf explants, respectively, spontaneously developed into multiple plantlets on the same MS medium (supplemented with 20 mg/L TDZ) for induction of RTBs, whereas >95.00% of in vitro RTBs from each kind of explant developed into multiple plantlets on MS medium supplemented with 5.0 mg/L 6-benzylaminopurine (BAP). Morphological and histological analyses revealed that RTB is a novel type of SE structure that develops from the cortex cells of rhizoids. PMID:25744384

  6. Fluorescence fluctuation analysis of Arabidopsis thaliana somatic embryogenesis receptor-like kinase and brassinosteroid insensitive 1 receptor oligomerization.

    PubMed

    Hink, Mark A; Shah, Khalid; Russinova, Eugenia; de Vries, Sacco C; Visser, Antonie J W G

    2008-02-01

    Receptor kinases play a key role in the cellular perception of signals. To verify models for receptor activation through dimerization, an experimental system is required to determine the precise oligomerization status of proteins within living cells. Here we show that photon counting histogram analysis and dual-color fluorescence cross correlation spectroscopy are able to monitor fluorescently labeled proteins at the single-molecule detection level in living plant cells. In-frame fusion proteins of the brassinosteroid insensitive 1 (BRI1) receptor and the Arabidopsis thaliana somatic embryogenesis receptor-like kinases 1 and 3 (AtSERK1 and 3) to the enhanced cyan or yellow fluorescent protein were transiently expressed in plant cells. Although no oligomeric structures were detected for AtSERK3, 15% (AtSERK1) to 20% (BRI1) of the labeled proteins in the plasma membrane was found to be present as homodimers, whereas no evidence was found for higher oligomeric complexes. PMID:17905839

  7. Increased Putrescine Biosynthesis through Transfer of Mouse Ornithine Decarboxylase cDNA in Carrot Promotes Somatic Embryogenesis.

    PubMed Central

    Bastola, D. R.; Minocha, S. C.

    1995-01-01

    Carrot (Daucus carota L.) cells were transformed with Agrobacterium tumefaciens strains containing 3[prime]-truncated mouse ornithine decarboxylase (ODC) cDNA under the control of a cauliflower mosaic virus 35S promoter. A neomycin phosphotransferase gene linked with a nopaline synthase promoter was used to select transformed cell lines on kanamycin. Although the nontransformed cells contained no ODC, high amounts of mouse-specific ODC activity were observed in the transformed cells. Transgenic cells showed a significant increase in the cellular content of putrescine compared to control cells. Spermidine, however, remained unaffected. Not only did the transformed cells exhibit improved somatic embryogenesis in the auxin-free medium, they also regenerated some embryos in the presence of inhibitory concentrations of 2,4-dichlorophenoxyacetic acid. These cells acquired tolerance to [alpha]-difluoromethylarginine (a potent inhibitor of arginine decarboxylase) at concentrations that inhibit growth as well as embryogenesis in nontransformed carrot cells, showing that the mouse ODC can replace the carrot arginine decarboxylase for putrescine biosynthesis in the transgenic cells. PMID:12228581

  8. NORMALIZING SWEET ORANGE (C. SINENSIS (L.) OSBECK) SOMATIC EMBRYOGENESIS WITH SEMIPERMEABLE MEMBRANES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Development of citrus somatic embryos initiated from embryogenic callus generally results in abnormal morphologies during growth and development. Shoots can be regenerated by organogenesis from these abnormal structures, excised and rooted to recover plants. To normalize development, glycerol-indu...

  9. Characterization and expression analysis of SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) genes in sexual and apomictic Paspalum notatum.

    PubMed

    Podio, Maricel; Felitti, Silvina Andrea; Siena, Lorena Adelina; Delgado, Luciana; Mancini, Micaela; Seijo, José Guillermo; González, Ana María; Pessino, Silvina Claudia; Ortiz, Juan Pablo A

    2014-03-01

    The SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) gene plays a fundamental role in somatic embryogenesis of angiosperms, and is associated with apomixis in Poa pratensis. The objective of this work was to isolate, characterize and analyze the expression patterns of SERK genes in apomictic and sexual genotypes of Paspalum notatum. A conserved 200-bp gene fragment was amplified from genomic DNA with heterologous primers, and used to initiate a chromosomal walking strategy for cloning the complete sequence. This procedure allowed the isolation of two members of the P. notatum SERK family; PnSERK1, which is similar to PpSERK1, and PnSERK2, which is similar to ZmSERK2 and AtSERK1. Phylogenetic analyses indicated that PnSERK1 and PnSERK2 represent paralogous sequences. Southern-blot hybridization indicated the presence of at least three copies of SERK genes in the species. qRT-PCR analyses revealed that PnSERK2 was expressed at significantly higher levels than PnSERK1 in roots, leaves, reproductive tissues and embryogenic calli. Moreover, in situ hybridization experiments revealed that PnSERK2 displayed a spatially and chronologically altered expression pattern in reproductive organs of the apomictic genotype with respect to the sexual one. PnSERK2 is expressed in nucellar cells of the apomictic genotype at meiosis, but only in the megaspore mother cell in the sexual genotype. Therefore, apomixis onset in P. notatum seems to be correlated with the expression of PnSERK2 in nucellar tissue. PMID:24146222

  10. Characterization of VvSERK1, VvSERK2, VvSERK3 and VvL1L genes and their expression during somatic embryogenesis of grapevine (Vitis vinifera L.).

    PubMed

    Schellenbaum, Paul; Jacques, Alban; Maillot, Pascale; Bertsch, Christophe; Mazet, Flore; Farine, Sibylle; Walter, Bernard

    2008-12-01

    Little is known about the genes expressed during grapevine somatic embryogenesis. Both groups of Somatic Embryogenesis Receptor Kinase (SERK) and Leafy Cotyledon (LEC and L1L) genes seem to play key roles during somatic embryogenesis in various plant species. Therefore, we identified and analysed the sequences of VvSERK and VvL1L (Leafy cotyledon1-Like) genes. The deduced amino acid sequences of VvSERK1, VvSERK2 and VvSERK3 are very similar to that of registered SERK proteins, with highest homologies for the kinase domain in the C-terminal region. The amino acid sequence of VvL1L presents all the domains that are characteristic for LEC1 and L1L proteins, particularly, the 16 amino acid residues that serve as signature of the B-domain. Phylogenetic analysis distinguishes members of subclass LEC1 and subclass L1L, and VvL1L is closely related to L1L proteins. Using semi-quantitative RT-PCR, we studied gene expression of VvSERK1, VvSERK2, VvSERK3 and VvL1L in calli and somatic embryos obtained from anther culture of Vitis vinifera L. cv Chardonnay. Expression of VvSERK2 is relatively stable during in vitro culture. In contrast, VvSERK1, VvSERK3 and VvL1L are expressed more 4 to 6 weeks after transfer of the calli onto embryo induction medium, before the visible appearance of embryos on the calli as seen by environmental scanning electron microscopy. Later on (8 weeks after transfer) VvSERK1 expression is maintained in the embryogenic calli and VvSERK3 in the embryos, whereas VvL1L expression is very low. All together, these data suggest the involvement of VvSERK and VvL1L genes in grapevine somatic embryogenesis. PMID:18766346

  11. Establishment of embryonic cultures and somatic embryogenesis in callus culture of guggul-Commiphora wightii (Arnott.) Bhandari.

    PubMed

    Kumar, Sandeep; Suri, S S; Sonie, K C; Ramawat, K G

    2003-01-01

    Somatic embryogenesis in callus cultures of Commiphora wightii (Arnott.) Bhandari was achieved. Though the frequency of explants producing embryonic culture was low, immature zygotic embryos were the only suitable explants to produce embryonic callus after reciprocal transfers on media containing 2,4,5-trichlorophenoxy acetic acid (0.1 mgl(-1)) and kinetin (0.1 mgl(-1)) or devoid of growth regulators. All other media failed to produce embryonic callus. Embryonic cells were small, densely filled with cytoplasm and isodiametric as compared to non-embryonic cells, which were large, elongated and vacuolated. Maximum growth of embryonic callus was recorded on modified MS medium (MS-2 medium) supplemented with BA (0.25 mgl(-1)) and IBA (0.1 mgl(-1)). MS-2 salts supported higher growth of callus as compared to tissues grown on B5 medium containing same concentrations of plant growth regulators. Exogenous medium nutrients had no effect on somatic embryo development whereas plant growth regulators had little effect. Asynchronously growing embryos formed plantlets regularly which were successfully transferred to the field conditions. PMID:15267139

  12. Identification of a PTC-containing DlRan transcript and its differential expression during somatic embryogenesis in Dimocarpus longan.

    PubMed

    Fang, Zhi-Zhen; Lai, Cheng-Chun; Zhang, Ya-Ling; Lin, Yu-Ling; Lai, Zhong-Xiong

    2013-10-15

    RAN (Ras-related nuclear protein) plays crucial roles in multiple cellular processes in yeast, animals and plants. Here we present a DlRan gene and its alternative splicing transcripts containing premature terminator codons (PTCs), identified from embryogenic cultures in longan. Multiple alignment and splicing pattern analyses indicated that DlRan-1 transcript harboring PTC was the consequence of alternative splicing. The accumulation of DlRan PTC-containing transcripts increased significantly when the embryogenic calli were treated with the translation inhibitor, cycloheximide, indicating that DlRan-1 may be targeted by NMD. The analysis of expression profiles of DlRan transcripts revealed that differential expression levels of the alternative spliced DlRan transcripts occurred during the development of embryogenic callus, globular-shaped embryos, and cotyledon-shaped embryos, respectively, in the longan somatic embryogenesis, and were in consistent with the embryo development in corresponding wild-type transcripts. The present work offers evidence to speculate that the alternatively spliced PTC-containing transcripts can be functional and may shed light on expression regulation of DlRan during development of the longan somatic embryos. PMID:23933275

  13. Transcriptome analysis during somatic embryogenesis of the tropical monocot Elaeis guineensis: evidence for conserved gene functions in early development.

    PubMed

    Lin, Hsiang-Chun; Morcillo, Fabienne; Dussert, Stéphane; Tranchant-Dubreuil, Christine; Tregear, James W; Tranbarger, Timothy John

    2009-05-01

    With the aim of understanding the molecular mechanisms underlying somatic embryogenesis (SE) in oil palm, we examined transcriptome changes that occur when embryogenic suspension cells are initiated to develop somatic embryos. Two reciprocal suppression subtractive hybridization (SSH) libraries were constructed from oil palm embryogenic cell suspensions: one in which embryo development was blocked by the presence of the synthetic auxin analogue 2,4-dichlorophenoxyacetic acid (2,4-D: ) in the medium (proliferation library); and another in which cells were stimulated to form embryos by the removal of 2,4-D: from the medium (initiation library). A total of 1867 Expressed Sequence Tags (ESTs) consisting of 1567 potential unigenes were assembled from the two libraries. Functional annotation indicated that 928 of the ESTs correspond to proteins that have either no similarity to sequences in public databases or are of unknown function. Gene Ontology (GO) terms assigned to the two EST populations give clues to the underlying molecular functions, biological processes and cellular components involved in the initiation of embryo development. Macroarrays were used for transcript profiling the ESTs during SE. Hierarchical cluster analysis of differential transcript accumulation revealed 4 distinct profiles containing a total of 192 statistically significant developmentally regulated transcripts. Similarities and differences between the global results obtained with in vitro systems from dicots, monocots and gymnosperms will be discussed. PMID:19199047

  14. Yield performance and bean quality traits of cacao propagated by somatic embryogenesis and grafting

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Twelve cacao (Theobroma cacao) clones propagated by grafting and rooted cuttings of somatic embryo-derived plants were grown on an Ultisol soil at Corozal, Puerto Rico and evaluated for six years under intensive management. Year, variety, the year x variety and propagation treatment x variety intera...

  15. Clustering of Microarray Data Reveals Transcript Patterns Associated with Somatic Embryogenesis in Soybean1[w

    PubMed Central

    Thibaud-Nissen, Françoise; Shealy, Robin T.; Khanna, Anupama; Vodkin, Lila O.

    2003-01-01

    Globular somatic embryos can be induced from immature cotyledons of soybean (Glycine max L. Merr. cv Jack) placed on high levels of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryos develop from the adaxial side of the cotyledon, whereas the abaxial side evolves into a callus. Using a 9,280-cDNA clone array, we have compared steady-state RNA from the adaxial side from which embryos develop and from the abaxial callus at five time points over the course of the 4 weeks necessary for the development of globular embryos. In a second set of experiments, we have profiled the expression of each clone in the adaxial side during the same period. A total of 495 genes differentially expressed in at least one of these experiments were grouped according to the similarity of their expression profiles using a nonhierarchical clustering algorithm. Our results indicate that the appearance of somatic embryos is preceded by dedifferentiation of the cotyledon during the first 2 weeks on auxin. Changes in mRNA abundance of genes characteristic of oxidative stress and genes indicative of cell division in the adaxial side of the cotyledons suggest that the arrangement of the new cells into organized structures might depend on a genetically controlled balance between cell proliferation and cell death. Our data also suggest that the formation of somatic globular embryos is accompanied by the transcription of storage proteins and the synthesis of gibberellic acid. PMID:12746518

  16. Cellular and molecular changes associated with competence acquisition during passion fruit somatic embryogenesis: ultrastructural characterization and analysis of SERK gene expression.

    PubMed

    Rocha, Diego Ismael; Pinto, Daniela Lopes Paim; Vieira, Lorena Melo; Tanaka, Francisco André Ossamu; Dornelas, Marcelo Carnier; Otoni, Wagner Campos

    2016-03-01

    The integration of cellular and molecular data is essential for understanding the mechanisms involved in the acquisition of competence by plant somatic cells and the cytological changes that underlie this process. In the present study, we investigated the dynamics and fate of Passiflora edulis Sims cotyledon explants that were committed to somatic embryogenesis by characterizing the associated ultrastructural events and analysing the expression of a putative P. edulis ortholog of the Somatic Embryogenesis Receptor-like Kinase (SERK) gene. Embryogenic calli were obtained from zygotic embryo explants cultured on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Callus formation was initiated by the division of cells derived from the protodermal and subprotodermal cells on the abaxial side of the cotyledons. The isodiametric protodermal cells of the cotyledon explants adopted a columnar shape and became meristematic at the onset of PeSERK expression, which was not initially detected in explant cells. Therefore, we propose that these changes represent the first observable steps towards the acquisition of a competent state within this regeneration system. PeSERK expression was limited to the early stages of somatic embryogenesis; the expression of this gene was confined to proembryogenic zones and was absent in the embryos after the globular stage. Our data also demonstrated that the dynamics of the mobilization of reserve compounds correlated with the differentiation of the embryogenic callus. PMID:26008651

  17. Transcript profiling reveals complex auxin signalling pathway and transcription regulation involved in dedifferentiation and redifferentiation during somatic embryogenesis in cotton

    PubMed Central

    2012-01-01

    Background Somatic embryogenesis (SE), by which somatic cells of higher plants can dedifferentiate and reorganize into new plants, is a notable illustration of cell totipotency. However, the precise molecular mechanisms regulating SE remain unclear. To characterize the molecular events of this unique process, transcriptome analysis, in combination with biochemical and histological approaches, were conducted in cotton, a typical plant species in SE. Genome-wide profiling of gene expression allowed the identification of novel molecular markers characteristic of this developmental process. Results RNA-Seq was used to identify 5,076 differentially expressed genes during cotton SE. Expression profile and functional assignments of these genes indicated significant transcriptional complexity during this process, associated with morphological, histological changes and endogenous indole-3-acetic acid (IAA) alteration. Bioinformatics analysis showed that the genes were enriched for basic processes such as metabolic pathways and biosynthesis of secondary metabolites. Unigenes were abundant for the functions of protein binding and hydrolase activity. Transcription factor–encoding genes were found to be differentially regulated during SE. The complex pathways of auxin abundance, transport and response with differentially regulated genes revealed that the auxin-related transcripts belonged to IAA biosynthesis, indole-3-butyric acid (IBA) metabolism, IAA conjugate metabolism, auxin transport, auxin-responsive protein/indoleacetic acid-induced protein (Aux/IAA), auxin response factor (ARF), small auxin-up RNA (SAUR), Aux/IAA degradation, and other auxin-related proteins, which allow an intricate system of auxin utilization to achieve multiple purposes in SE. Quantitative real-time PCR (qRT-PCR) was performed on selected genes with different expression patterns and functional assignments were made to demonstrate the utility of RNA-Seq for gene expression profiles during cotton SE. Conclusion We report here the first comprehensive analysis of transcriptome dynamics that may serve as a gene expression profile blueprint in cotton SE. Our main goal was to adapt the RNA-Seq technology to this notable development process and to analyse the gene expression profile. Complex auxin signalling pathway and transcription regulation were highlighted. Together with biochemical and histological approaches, this study provides comprehensive gene expression data sets for cotton SE that serve as an important platform resource for further functional studies in plant embryogenesis. PMID:22817809

  18. High expression of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE coincides with initiation of various developmental pathways in in vitro culture of Trifolium nigrescens.

    PubMed

    Pilarska, Maria; Malec, Przemysław; Salaj, Jan; Bartnicki, Filip; Konieczny, Robert

    2016-03-01

    The aim of this study was to identify and examine the expression pattern of the ortholog of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE gene from Trifolium nigrescens (TnSERK) in embryogenic and non-regenerative cultures of immature cotyledonary-stage zygotic embryos (CsZEs). In the presence of 1-naphthaleneacetic acid and N(6)-[2-isopentenyl]-adenine, the CsZE regenerated embryoids directly and in a lengthy culture produced callus which was embryogenic or remained non-regenerative. As revealed by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), the TnSERK was expressed in both embryogenic and non-regenerative cultures, but the expression level was significantly higher in embryogenic ones. An in situ RNA hybridization assay revealed that the expression of TnSERK preceded the induction of cell division in explants, and then, it was maintained exclusively in actively dividing cells from which embryoids, embryo-like structures (ELSs), callus or tracheary elements were produced. However, the cells involved in different morphogenic events differed in intensity of hybridization signal which was the highest in embryogenic cells. The TnSERK was up-regulated during the development of embryoids, but in cotyledonary embryos, it was preferentially expressed in the regions of the apical meristems. The occurrence of morphological and anatomical abnormalities in embryoid development was preceded by a decline in TnSERK expression, and this coincided with the parenchymatization of the ground tissue in developing ELSs. TnSERK was also down-regulated during the maturation of parenchyma and xylem elements in CsZE and callus. Altogether, these data suggest the involvement of TnSERK in the induction of various developmental programs related to differentiation/transdifferentiation and totipotent state of cell(s). PMID:25876517

  19. Somatic embryogenesis in protoplast derived calli of cultivated jute, Corchorus capsularis L.

    PubMed

    Saha, T; Sen, S K

    1992-01-01

    Protoplasts were isolated from cotyledon, hypocotyl and mesophyll cells of Corchorus capsularis L., a major fibre crop, by one step enzyme digestion method. They were further cultured successfully on modified KM-8p (Kao and Michayluk, 1975) medium to form microcalli. The required cultural conditions could be used to achieve 34% to 78% plating efficiency, depending upon the source of protoplasts. Hypocotyl protoplasts gave the highest plating efficiency. On transfer to regeneration medium, somatic embryos developed at high frequency. The present success is a significant step forward in the development of meaningful plant cell culture methods for application in jute. PMID:24212878

  20. The union of somatic gonad precursors and primordial germ cells during C. elegans embryogenesis

    PubMed Central

    Rohrschneider, Monica R.; Nance, Jeremy

    2013-01-01

    Somatic gonadal niche cells control the survival, differentiation, and proliferation of germline stem cells. The establishment of this niche-stem cell relationship is critical, and yet the precursors to these two cell types are often born at a distance from one another. The simple C. elegans gonadal primordium, which contains two somatic gonad precursors (SGPs) and two primordial germ cells (PGCs), provides an accessible model for determining how stem cell and niche cell precursors first assemble during development. To visualize the morphogenetic events that lead to formation of the gonadal primordium, we generated transgenic strains to label the cell membranes of the SGPs and PGCs and captured time-lapse movies as the gonadal primordium formed. We identify three distinct phases of SGP behavior: posterior migration along the endoderm towards the PGCs, extension of a single long projection around the adjacent PGC, and a dramatic wrapping over the PGC surfaces. We show that the endoderm and PGCs are dispensable for SGP posterior migration and initiation of projections. However, both tissues are required for the final positioning of the SGPs and the morphology of their projections, and PGCs are absolutely required for SGP wrapping behaviors. Finally, we demonstrate that the basement membrane component laminin, which localizes adjacent to the developing gonadal primordium, is required to prevent the SGPs from over-extending past the PGCs. Our findings provide a foundation for understanding the cellular and molecular regulation of the establishment of a niche-stem cell relationship. PMID:23562590

  1. Somatic embryogenesis and plant regeneration in tissue cultures of radish (Raphanus sativus L.).

    PubMed

    Jeong, W J; Min, S R; Liu, J R

    1995-07-01

    Hypocotyl segments of 2- to 3-week-old radish (Raphanus sativus L. cv. F1 Handsome Fall) seedlings produced yellowish compact calli when cultured on Murashige and Skoog's (MS) medium supplemented with 1 mgl(-1) 2,4-dichlorophenoxyacetic acid (2,4-D). Upon transfer onto medium containing 6-benzyladenine and α-naphthaleneacetic acid, up to 5.3% of the calli gave rise to a few somatic embryos. When subcultured for 3 to 6 months, 7% of the yellowish, compact calli produced white, compact calli which formed numerous embryos. These calli maintained their embryogenic capacity for over 18 months. When cultured on medium containing 0.1 to 3 mgl(-1) 2,4-D, up to 90% of longitudinally sliced somatic embryo halves produced calli with numerous secondary embryos. Embryos were transferred onto medium containing 0.1 mgl(-1) 2,4-D and 1 mgl(-1) abscisic acid where they developed into the cotyledonary stage. Upon transfer onto half-strength MS basal medium, approximately 90% of the embryos developed into plantlets. These plantlets were successfully transplanted in potting soil and after cold treatment they were grown to maturity in a phytotron. PMID:24194314

  2. Somatic embryogenesis and plant regeneration in diploid Allium fistulosum × A. cepa F1 hybrid onions.

    PubMed

    Lu, C C; Currah, L; Peffley, E B

    1989-03-01

    Procedures were developed for disinfestation of non-dormant basal plate tissue excised from field grown basal plate tissue of diploid Allium fistulosum × A. cepa F1 hybrid onions. Contamination levels varied with the season and vegetative development of plant material. Callus initiated from basal plate tissue and immature inflorescences of the F1 hybrids was maintained on a BDS-based medium containing 0.75 mg/l picloram and 2.0 mg/l BA. When this medium was supplemented with vitamins and glycine, and with proline at 2.5 gm/1, somatic embryos began to form. Their development continued on a BDS-based shoot promotion medium containing 0.03 mg/l picloram and 0.32 mg/l 2iP supplemented with vitamins, glycine and proline. Genotypes differed significantly in the numbers of structures regenerated. Plantlets from somatic embryos were rooted into BDS or half-strength BDS medium without growth substances and were successfully transferred to sterilized potting mix in plastic commercial corsage boxes. PMID:24240465

  3. Somatic embryogenesis and plant regeneration from protoplast culture of Crocus pallasii subsp. haussknechtii.

    PubMed

    Karamian, Roya

    2007-02-15

    A protocol has been developed for plant regeneration from protoplast culture of Crocus pallasii subsp. haussknechtii using regenerable embryogenic calli obtained from shoot meristem culture on MS+9.28 microM kinetin+4.52 microM 2,4-D. Protoplasts were isolated directly from embryogenic calli, embedded in Ca-alginate beads and cultured with nurse cells in MS+4.64 microM kinetin+4.52 microM 2,4-D+5.68 microM ascorbic acid+0.3 M mannitol at 20 +/- 2 degrees C in darkness. After appearing ofmicrocalli on the surface of the beads, they were transferred onto 1/2MS+2.32 microM kinetin+2.26 microM 2,4-D+5.68 microM ascorbic acid for growth of embryogenic calli. Somatic embryos matured on MS medium growth regulator free and germinated on 1/2MS+14.45 microM GA3 +4.43 microM BA at 20 +/- 2 degrees C in a 16/8 h light/dark cycle. PMID:19069554

  4. Genetic instability in calamondin (Citrus madurensis Lour.) plants derived from somatic embryogenesis induced by diphenylurea derivatives.

    PubMed

    Siragusa, Mirko; Carra, Angela; Salvia, Lidia; Puglia, Anna Maria; De Pasquale, Fabio; Carimi, Francesco

    2007-08-01

    Somatic embryos were regenerated in vitro from calamondin style-stigma explants cultured in the presence of N (6)-benzylaminopurine (BAP) cytokinin and three synthetic phenylurea derivatives, N-(2-chloro-4-pyridyl)-N-phenylurea (4-CPPU), N-phenyl-N'-benzothiazol-6-ylurea (PBU) and N,N'-bis-(2,3-methilendioxyphenyl)urea (2,3-MDPU). The phenylurea derivative compounds tested at micromolar level (12 muM) were able to induce a percentage of responsive explants significantly higher from that obtained with BAP and hormone-free (HF) conditions. In order to verify the genetic stability of the regenerants, 27 plants coming from different embryogenic events were randomly selected from each different culture condition and evaluated for somaclonal variations using inter-simple sequence repeat and random amplified polymorphic DNA analyses. We observed that 2,3-MDPU and PBU gave 3.7% of somaclonal mutants, whereas 4-CPPU gave 7.4% of mutants. No somaclonal variability was observed when plantlets were regenerated in BAP or HF medium. Although diphenylurea derivatives show a higher embryogenic potential as compared to BAP, they induce higher levels of somaclonal variability. This finding should be taken in consideration when new protocols for clonal propagation are being developed. PMID:17333016

  5. Evolution of endogenous hormone concentration in embryogenic cultures of carrot during early expression of somatic embryogenesis.

    PubMed

    Jiménez, V M; Guevara, E; Herrera, J; Bangerth, F

    2005-01-01

    Embryogenic callus and suspension cultures of carrot (Daucus carota L., cv. Nantaise), growing on/in medium including 1 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D), were transferred to medium with or without this plant growth regulator, to impair or induce, respectively, further development of somatic embryos. The endogenous hormone levels of the cultures were determined over 7 days by means of radio-immunoassay, to characterize their evolution in the initial stages of embryo development. In general, levels of indoleacetic acid (IAA) and abscisic acid (ABA) showed only short-lived differences among treatments during this time in both types of tissue analyzed (i.e., a peak of IAA in callus cultures in the absence of 2,4-D, 48 h after medium change, and higher ABA contents 144 h after subculture of suspension cultures in the presence of 2,4-D). Gibberellins (1, 3 and 20) were detected only in suspension cultures devoid of 2,4-D, starting 24 h after subculture. Concerning the evaluated cytokinins-zeatin/zeatin riboside and N6(Delta2-isopentenyl) adenine/N6(Delta2-isopentenyl) adenosine-the most remarkable observation is that high levels of the former generally coincided with low concentrations of the latter, indicating a shift from precursor to the active form, and vice versa. PMID:15375630

  6. A novel in vitro protocol for inducing direct somatic embryogenesis in Phalaenopsis aphrodite without taking explants.

    PubMed

    Feng, Jia-Hua; Chen, Jen-Tsung

    2014-01-01

    An alternative in vitro protocol for embryo induction directly from intact living seedlings of Phalaenopsis aphrodite subspecies formosana was established in this study. Without the supplementation of plant growth regulators (PGRs), no embryos were obtained from all the seedlings when cultured on the solid medium. In contrast, embryos formed from the seedlings on the 2-layer medium and the 2-step culture system without the use of PGRs. It was found that the age of the seedlings affected embryo induction. The 2-month-old seedlings typically had higher embryogenic responses when compared with the 4-month-old seedlings in the 2-layer medium or 2-step system. For the 2-month-old seedlings, 1 mg/L TDZ resulted in the highest number of embryos at the distal site of the shoot. However, on the leaves' surface, 0.5 mg/L TDZ induced the highest number of embryos. When the 2-month-old seedlings were cultured using the 2-step method at 1 mg/L of TDZ, the highest embryogenic response was obtained, with an average of 44 embryos formed on each seedling. These adventitious embryos were able to convert into plantlets in a PGR-free 1/2 MS medium, and the plantlets had normal morphology and growth. PMID:24963505

  7. A Novel In Vitro Protocol for Inducing Direct Somatic Embryogenesis in Phalaenopsis aphrodite without Taking Explants

    PubMed Central

    Chen, Jen-Tsung

    2014-01-01

    An alternative in vitro protocol for embryo induction directly from intact living seedlings of Phalaenopsis aphrodite subspecies formosana was established in this study. Without the supplementation of plant growth regulators (PGRs), no embryos were obtained from all the seedlings when cultured on the solid medium. In contrast, embryos formed from the seedlings on the 2-layer medium and the 2-step culture system without the use of PGRs. It was found that the age of the seedlings affected embryo induction. The 2-month-old seedlings typically had higher embryogenic responses when compared with the 4-month-old seedlings in the 2-layer medium or 2-step system. For the 2-month-old seedlings, 1 mg/L TDZ resulted in the highest number of embryos at the distal site of the shoot. However, on the leaves' surface, 0.5 mg/L TDZ induced the highest number of embryos. When the 2-month-old seedlings were cultured using the 2-step method at 1 mg/L of TDZ, the highest embryogenic response was obtained, with an average of 44 embryos formed on each seedling. These adventitious embryos were able to convert into plantlets in a PGR-free 1/2 MS medium, and the plantlets had normal morphology and growth. PMID:24963505

  8. Somatic embryogenesis, tetraploidy, and variant leaf morphology in transgenic diploid strawberry (Fragaria vesca subspecies vesca ‘Hawaii 4’)

    PubMed Central

    2014-01-01

    Background The diploid (2n = 2x = 14) strawberry model plant Fragaria vesca ssp. vesca ‘Hawaii 4’ was employed for functional analysis of expressed DNA sequences initially identified as being unique to Fragaria and of unknown or poorly understood function. ‘Hawaii 4’ is prominent in strawberry research due to its ease of Agrobacterium-mediated transformation and regenerability, and its status as the source of the first complete strawberry genomic sequence. Our studies of a set of transformants have documented intriguing, construct-associated effects on leaf morphology, and provide important and unexpected insights into the performance of the ‘Hawaii 4’ transformation and regeneration system. Results Following Agrobacterium-mediated transformation of leaf explants with gene constructs carried by Gateway® vectors, plants were regenerated using a modified version of an established ‘Hawaii 4’ protocol. Expanding upon the findings of prior studies, we documented that plantlet regeneration was occurring via a somatic embryogenic rather than an organogenic developmental pathway. Among transformants, several variations in leaf morphology were observed. Unexpectedly, a particular leaf variant type, occurring in ~17% of all regenerants independent of construct type, was found to be attributable to tetraploidy. The tetraploidy-associated alteration in leaf morphology could be differentiated from the leaf morphology of diploid regenerants on the basis of a quantitative ratio of leaf dimensions: B/A, where B is the width of the central leaflet and A is the overall width of the trifoliate leaf. Variant effects on leaf morphology of four different transgenic constructs were also documented, and were in all cases distinguishable from the effects of tetraploidy. Conclusions These results define opportunities to optimize the existing ‘Hawaii 4’ protocol by focusing on treatments that specifically promote somatic embryogenesis. The reported morphological metric and descriptions will guide future transgenic studies using the ‘Hawaii 4’ model system by alerting researchers to the potential occurrence of polyploid regenerants, and to differentiating the effects on leaf morphology due to polyploidy versus transgenic manipulations. Finally, an intriguing spectrum of leaf morphology alterations resulting from manipulation of expressed sequences of uncertain function is documented, providing a foundation for detailed studies of the respective genes and their functional roles. PMID:24418064

  9. Alterations in the Transcriptome of Soybean in Response to Enhanced Somatic Embryogenesis Promoted by Orthologs of AGAMOUS-Like15 and AGAMOUS-Like181[C][W][OPEN

    PubMed Central

    Zheng, Qiaolin; Perry, Sharyn E.

    2014-01-01

    Somatic embryogenesis (SE) is a poorly understood process during which competent cells respond to inducing conditions, allowing the development of somatic embryos. It is important for the regeneration of transgenic plants, including for soybean (Glycine max). We report here that constitutive expression of soybean orthologs of the Arabidopsis (Arabidopsis thaliana) MADS box genes AGAMOUS-Like15 (GmAGL15) and GmAGL18 increased embryogenic competence of explants from these transgenic soybean plants. To understand how GmAGL15 promotes SE, expression studies were performed. Particular genes of interest involved in embryogenesis (ABSCISIC ACID-INSENSITIVE3 and FUSCA3) were found to be directly up-regulated by GmAGL15 by using a combination of quantitative reverse transcription-polymerase chain reaction and chromatin immunoprecipitation. To look more broadly at changes in gene expression in response to GmAGL15, we assessed the transcriptome using the Affymetrix Soybean Genome Array. Interestingly, the gene expression profile of 35Spro:GmAGL15 explants (0 d in culture) was found to resemble nontransgenic tissue that had been induced for SE by being placed on induction medium for 3 d, possibly explaining the more rapid SE development observed on 35Spro:GmAGL15 tissue. In particular, transcripts from genes related to the stress response showed increased transcript accumulation in explants from 35Spro:GmAGL15 tissue. These same genes also showed increased transcript accumulation in response to culturing nontransgenic soybean explants on the medium used to induce SE. Overexpression of GmAGL15 may enhance SE by making the tissue more competent to respond to 2,4-dichlorophenoxyacetic acid induction by differential regulation of genes such as those involved in the stress response, resulting in more rapid and prolific SE. PMID:24481137

  10. LEAFY COTYLEDON1-CASEIN KINASE I-TCP15-PHYTOCHROME INTERACTING FACTOR4 Network Regulates Somatic Embryogenesis by Regulating Auxin Homeostasis1[OPEN

    PubMed Central

    Min, Ling; Hu, Qin; Li, Yaoyao; Xu, Jiao; Ma, Yizan; Zhu, Longfu; Yang, Xiyan; Zhang, Xianlong

    2015-01-01

    Somatic embryogenesis (SE) is an efficient tool for the propagation of plant species and also, a useful model for studying the regulatory networks in embryo development. However, the regulatory networks underlying the transition from nonembryogenic callus to somatic embryos during SE remain poorly understood. Here, we describe an upland cotton (Gossypium hirsutum) CASEIN KINASE I gene, GhCKI, which is a unique key regulatory factor that strongly affects SE. Overexpressing GhCKI halted the formation of embryoids and plant regeneration because of a block in the transition from nonembryogenic callus to somatic embryos. In contrast, defective GhCKI in plants facilitated SE. To better understand the mechanism by which GhCKI regulates SE, the regulatory network was analyzed. A direct upstream negative regulator protein, cotton LEAFY COTYLEDON1, was identified to be targeted to a cis-element, CTTTTC, in the promoter of GhCKI. Moreover, GhCKI interacted with and phosphorylated cotton CINCINNATA-like TEOSINTE BRANCHED1-CYCLOIDEA-PCF transcription factor15 by coordinately regulating the expression of cotton PHYTOCHROME INTERACTING FACTOR4, finally disrupting auxin homeostasis, which led to increased cell proliferation and aborted somatic embryo formation in GhCKI-overexpressing somatic cells. Our results show a complex process of SE that is negatively regulated by GhCKI through a complex regulatory network. PMID:26491146

  11. Hormonally regulated overexpression of Arabidopsis WUS and conifer LEC1 (CHAP3A) in transgenic white spruce: implications for somatic embryo development and somatic seedling growth.

    PubMed

    Klimaszewska, Krystyna; Pelletier, Gervais; Overton, Catherine; Stewart, Don; Rutledge, Robert G

    2010-07-01

    Adult conifers are still recalcitrant in clonal propagation despite significant advances in forest tree biotechnology. Plant regeneration through somatic embryogenesis from explants older than mature zygotic embryos is either difficult or impossible to achieve. To investigate if ectopic expression of transcription factors involved in the induction of the embryogenic process would induce somatic embryogenesis in Picea glauca (white spruce) somatic plants, we used the LEAFY-COTYLEDON1 homolog cloned from Picea mariana, CHAP3A, and Arabidopsis thaliana WUS to transform embryonal mass of P. glauca. Ectopic gene expression was induced by 17-beta-estradiol during stages of somatic embryogenesis (early embryogenesis and late embryogenesis) and somatic seedling growth in the transgenics. Of the two transcription factors, only WUS produced severe phenotypes by disrupting the development of somatic embryos on the maturation medium and inhibiting germination. However, none of the transgenes induced ectopic somatic embryogenesis even in the presence of plant growth regulators. Absolute quantitative PCR confirmed the expression of both CHAP3A and WUS in transgenic embryonal mass and in all parts of somatic seedlings. A high expression of the transgenes did not influence expression profiles of any of the ten other transcription factors tested, some of which have been known to be involved in the process of embryogenesis. Implications of these results for further work are discussed. PMID:20424847

  12. Expression patterns of LmAP2L1 and LmAP2L2 encoding two-APETALA2 domain proteins during somatic embryogenesis and germination of hybrid larch (Larix x marschlinsii).

    PubMed

    Guillaumot, Damien; Lelu-Walter, Marie-Anne; Germot, Agns; Meytraud, Fanny; Gastinel, Louis; Riou-Khamlichi, Catherine

    2008-06-16

    Two APETALA2 domain transcription factors were characterized first in angiosperms, and, recently, in several gymnosperms. These proteins are involved in several processes, from flowering to embryogenesis in Arabidopsis thaliana. We extrapolated this result to hybrid larch (Larixxmarschlinsii Coaz) resulting from a cross between European (Larix decidua) and Japanese (Larix kaempferi) larches. Somatic embryogenesis is well described and controlled for this Pinaceae. We characterized two-AP2 domain genes: LmAP2L1 and LmAP2L2. Phylogenetic analysis confirmed that LmAP2L1 and LmAP2L2 were orthologous to Norway spruce PaAP2L1 and PaAP2L2 and that L1 forms appeared to be specific to Pinaceae. RT-PCR analysis showed that larch APETALA2 was differentially expressed during late somatic embryogenesis and during the first steps of germination. Whereas LmAP2L2 was constitutively expressed during this process, LmAP2L1 expression appeared only during late somatic embryogenesis, when embryos were able to germinate. Further, LmAP2L1 appeared to be the preferentially expressed form during embryo germination. Thus, LmAP2L1 seems to be a valuable molecular marker for hybrid larch late somatic embryogenesis and could play a role during post-embryonic development. PMID:18160178

  13. Conditions Favorable for the Somatic Embryogenesis in Carrot Cell Culture Enhance Expression of the roIC Promoter-GUS Fusion Gene 1

    PubMed Central

    Fujii, Nobuharu; Uchimiya, Hirofumi

    1991-01-01

    We obtained carrot (Daucus carota) cells possessing the 5′-noncoding sequence of the ORF12 gene (roIC) of TL-DNA of the Ri plasmid and a structural gene of bacterial β-glucuronidase by Agrobacterium-mediated transformation. When such cells were cultured in medium containing 2,4-dichlorophenoxyacetic acid, substantial reduction in β-glucuronidase activity was observed. Upon transferring the cells from a 2,4-D-containing medium to one devoid of 2,4-dichlorophenoxyacetic acid, enhanced expression of β-glucuronidase in somatic embryo development was recorded. Activation by gibberillic acid and suppression by abscisic acid of β-glucuronidase activities, in concord with embryogenesis, were also noted. Images Figure 2 PMID:16667958

  14. Conditions Favorable for the Somatic Embryogenesis in Carrot Cell Culture Enhance Expression of the roIC Promoter-GUS Fusion Gene.

    PubMed

    Fujii, N; Uchimiya, H

    1991-01-01

    We obtained carrot (Daucus carota) cells possessing the 5'-noncoding sequence of the ORF12 gene (roIC) of TL-DNA of the Ri plasmid and a structural gene of bacterial beta-glucuronidase by Agrobacterium-mediated transformation. When such cells were cultured in medium containing 2,4-dichlorophenoxyacetic acid, substantial reduction in beta-glucuronidase activity was observed. Upon transferring the cells from a 2,4-D-containing medium to one devoid of 2,4-dichlorophenoxyacetic acid, enhanced expression of beta-glucuronidase in somatic embryo development was recorded. Activation by gibberillic acid and suppression by abscisic acid of beta-glucuronidase activities, in concord with embryogenesis, were also noted. PMID:16667958

  15. INDUCTION OF SOMATIC EMBRYOGENESIS IN O'HENRY CULTIVAR OF PEACH (PRUNUS PERSICA)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The peach industry plays an important role in the agricultural economy of the southeastern United States, annually producing 35% of the American peach crop. Peach tree short life (PTSL) syndrome results in the drastic decline in peach tree population and orchard longevity. It prematurely kills trees...

  16. Formulation of nutrient medium for in vitro somatic embryo induction in Plantago ovata forsk.

    PubMed

    Saha, Priyanka; Bandyopadhyay, Subhendu; Raychaudhuri, Sarmistha Sen

    2011-05-01

    A nutrient medium has been formulated by altering the macro- and micro-elemental concentration in the culture medium for in vitro somatic embryo induction of economically important medicinal plant Plantago ovata Forsk .A comparison was made between induced embryos with normal embryos (produced in Murashige and Skoog (MS) medium) to observe frequency of embryo induction and also to determine regeneration efficiency. In the present investigation, three different media have been formulated. Among them, FM3 (formulated media, treatment 3) was the most suitable for increasing the frequency of somatic embryo production and regeneration of P. ovata Forsk. Better result was obtained using formulated medium than with MS medium. PMID:20405339

  17. Comparative analysis reveals dynamic changes in miRNAs and their targets and expression during somatic embryogenesis in longan (Dimocarpus longan Lour.).

    PubMed

    Lin, Yuling; Lai, Zhongxiong

    2013-01-01

    Somatic embryogenesis (SE), which resembles zygotic embryogenesis, is an essential component of the process of plant cell differentiation and embryo development. Although microRNAs (miRNAs) are important regulators of many plant develop- mental processes, their roles in SE have not been thoroughly investigated. In this study, we used deep-sequencing, computational, and qPCR methods to identify, profile, and describe conserved and novel miRNAs involved in longan (Dimocarpus longan) SE. A total of 643 conserved and 29 novel miRNAs (including star strands) from more than 169 miRNA families were identified in longan embryogenic tissue using Solexa sequencing. By combining computational and degradome sequencing approaches, we were able to predict 2063 targets of 272 miRNAs and verify 862 targets of 181 miRNAs. Target annotation revealed that the putative targets were involved in a broad variety of biological processes, including plant metabolism, signal transduction, and stimulus response. Analysis of stage- and tissue-specific expressions of 20 conserved and 4 novel miRNAs indicated their possible roles in longan SE. These miRNAs were dlo-miR156 family members and dlo-miR166c* associated with early embryonic culture developmental stages; dlo-miR26, dlo-miR160a, and families dlo-miR159, dlo-miR390, and dlo-miR398b related to heart-shaped and torpedo- shaped embryo formation; dlo-miR4a, dlo-miR24, dlo-miR167a, dlo-miR168a*, dlo-miR397a, dlo-miR398b.1, dlo-miR398b.2, dlo-miR808 and dlo-miR5077 involved in cotyledonary embryonic development; and dlo-miR17 and dlo-miR2089*-1 that have regulatory roles during longan SE. In addition, dlo-miR167a, dlo-miR808, and dlo-miR5077 may be required for mature embryo formation. This study is the first reported investigation of longan SE involving large-scale cloning, characterization, and expression profiling of miRNAs and their targets. The reported results contribute to our knowledge of somatic embryo miRNAs and provide insights into miRNA biogenesis and expression in plant somatic embryo development. PMID:23593197

  18. Comparative Analysis Reveals Dynamic Changes in miRNAs and Their Targets and Expression during Somatic Embryogenesis in Longan (Dimocarpus longan Lour.)

    PubMed Central

    Lin, Yuling; Lai, Zhongxiong

    2013-01-01

    Somatic embryogenesis (SE), which resembles zygotic embryogenesis, is an essential component of the process of plant cell differentiation and embryo development. Although microRNAs (miRNAs) are important regulators of many plant develop- mental processes, their roles in SE have not been thoroughly investigated. In this study, we used deep-sequencing, computational, and qPCR methods to identify, profile, and describe conserved and novel miRNAs involved in longan (Dimocarpus longan) SE. A total of 643 conserved and 29 novel miRNAs (including star strands) from more than 169 miRNA families were identified in longan embryogenic tissue using Solexa sequencing. By combining computational and degradome sequencing approaches, we were able to predict 2063 targets of 272 miRNAs and verify 862 targets of 181 miRNAs. Target annotation revealed that the putative targets were involved in a broad variety of biological processes, including plant metabolism, signal transduction, and stimulus response. Analysis of stage- and tissue-specific expressions of 20 conserved and 4 novel miRNAs indicated their possible roles in longan SE. These miRNAs were dlo-miR156 family members and dlo-miR166c* associated with early embryonic culture developmental stages; dlo-miR26, dlo-miR160a, and families dlo-miR159, dlo-miR390, and dlo-miR398b related to heart-shaped and torpedo- shaped embryo formation; dlo-miR4a, dlo-miR24, dlo-miR167a, dlo-miR168a*, dlo-miR397a, dlo-miR398b.1, dlo-miR398b.2, dlo-miR808 and dlo-miR5077 involved in cotyledonary embryonic development; and dlo-miR17 and dlo-miR2089*-1 that have regulatory roles during longan SE. In addition, dlo-miR167a, dlo-miR808, and dlo-miR5077 may be required for mature embryo formation. This study is the first reported investigation of longan SE involving large-scale cloning, characterization, and expression profiling of miRNAs and their targets. The reported results contribute to our knowledge of somatic embryo miRNAs and provide insights into miRNA biogenesis and expression in plant somatic embryo development. PMID:23593197

  19. Factors influencing somatic embryogenesis, regeneration, and Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) cultivar TME14

    PubMed Central

    Nyaboga, Evans N.; Njiru, Joshua M.; Tripathi, Leena

    2015-01-01

    Routine production of large numbers of transgenic plants is required to fully exploit advances in cassava biotechnology and support development of improved germplasm for deployment to farmers. This article describes an improved, high-efficiency transformation protocol for recalcitrant cassava cultivar TME14 preferred in Africa. Factors that favor production of friable embryogenic calli (FEC) were found to be use of DKW medium, crushing of organized embryogenic structures (OES) through 1–2 mm sized metal wire mesh, washing of crushed OES tissues and short exposure of tyrosine to somatic embryos; and transformation efficiency was enhanced by use of low Agrobacterium density during co-cultivation, co-centrifugation of FEC with Agrobacterium, germination of paramomycin resistant somatic embryos on medium containing BAP with gradual increase in concentration and variations of the frequency of subculture of cotyledonary-stage embryos on shoot elongation medium. By applying the optimized parameters, FEC were produced for cassava cultivar TME14 and transformed using Agrobacterium strain LBA4404 harboring the binary vector pCAMBIA2301. About 70–80 independent transgenic lines per ml settled cell volume (SCV) of FEC were regenerated on selective medium. Histochemical GUS assays confirmed the expression of gusA gene in transformed calli, somatic embryos and transgenic plants. The presence and integration of the gusA gene were confirmed by PCR and Southern blot analysis, respectively. RT-PCR analysis of transgenic plants confirmed the expression of gusA gene. This protocol demonstrates significantly enhanced transformation efficiency over existing cassava transformation protocols and could become a powerful tool for functional genomics and transferring new traits into cassava. PMID:26113851

  20. Factors influencing somatic embryogenesis, regeneration, and Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) cultivar TME14.

    PubMed

    Nyaboga, Evans N; Njiru, Joshua M; Tripathi, Leena

    2015-01-01

    Routine production of large numbers of transgenic plants is required to fully exploit advances in cassava biotechnology and support development of improved germplasm for deployment to farmers. This article describes an improved, high-efficiency transformation protocol for recalcitrant cassava cultivar TME14 preferred in Africa. Factors that favor production of friable embryogenic calli (FEC) were found to be use of DKW medium, crushing of organized embryogenic structures (OES) through 1-2 mm sized metal wire mesh, washing of crushed OES tissues and short exposure of tyrosine to somatic embryos; and transformation efficiency was enhanced by use of low Agrobacterium density during co-cultivation, co-centrifugation of FEC with Agrobacterium, germination of paramomycin resistant somatic embryos on medium containing BAP with gradual increase in concentration and variations of the frequency of subculture of cotyledonary-stage embryos on shoot elongation medium. By applying the optimized parameters, FEC were produced for cassava cultivar TME14 and transformed using Agrobacterium strain LBA4404 harboring the binary vector pCAMBIA2301. About 70-80 independent transgenic lines per ml settled cell volume (SCV) of FEC were regenerated on selective medium. Histochemical GUS assays confirmed the expression of gusA gene in transformed calli, somatic embryos and transgenic plants. The presence and integration of the gusA gene were confirmed by PCR and Southern blot analysis, respectively. RT-PCR analysis of transgenic plants confirmed the expression of gusA gene. This protocol demonstrates significantly enhanced transformation efficiency over existing cassava transformation protocols and could become a powerful tool for functional genomics and transferring new traits into cassava. PMID:26113851

  1. Induction of Pluripotency in Mouse Somatic Cells with Lineage Specifiers

    PubMed Central

    Shu, Jian; Wu, Chen; Wu, Yetao; Li, Zhiyuan; Shao, Sida; Zhao, Wenhui; Tang, Xing; Yang, Huan; Shen, Lijun; Zuo, Xiaohan; Yang, Weifeng; Shi, Yan; Chi, Xiaochun; Zhang, Hongquan; Gao, Ge; Shu, Youmin; Yuan, Kehu; He, Weiwu; Tang, Chao; Zhao, Yang; Deng, Hongkui

    2014-01-01

    SUMMARY The reprogramming factors that induce pluripotency have been identified primarily from embryonic stem cell (ESC)-enriched, pluripotency-associated factors. Here we report that during mouse somatic cell reprogramming, pluripotency can be induced with lineage specifiers that are pluripotency rivals to suppress ESC identity, most of which are not enriched in ESCs. We found that OCT4 and SOX2, the core regulators of pluripotency, can be replaced by lineage specifiers that are involved in mesendodermal (ME) specification and in ectodermal (ECT) specification, respectively. OCT4 and its substitutes attenuated the elevated expression of a group of ECT genes whereas SOX2 and its substitutes curtailed a group of ME genes during reprogramming. Surprisingly, the two counteracting lineage specifiers can synergistically induce pluripotency in the absence of both OCT4 and SOX2. Our study suggests a “seesaw model,” in which a balance that is established using pluripotency factors and/or counteracting lineage specifiers can facilitate reprogramming. PMID:23706735

  2. Development of grapevine somatic embryogenesis using an air-lift bioreactor as an efficient tool in the generation of transgenic plants.

    PubMed

    Tapia, Eduardo; Sequeida, Alvaro; Castro, Alvaro; Montes, Christian; Zamora, Pablo; López, Reinaldo; Acevedo, Fernando; Prieto, Humberto

    2009-01-01

    The grapevine genetic transformation programs have relayed on the use of solid media-based somatic embryogenesis. To reach a high throughput of candidate gene evaluation in 'Thompson Seedless', a semi-automatic system allowing viable transformation of explants was designed. An intermediate procedure using liquid media and agitated flasks was first characterized, leading to reduction in the biomass duplication time of pro-embryogenic (PE) cells from 30 d in dishes to 14 d. The oxygen transfer coefficient value in this system was 213h(-1) at 120rpm and 25 degrees C with a 16/8-h (light/darkness) photoperiod. The scaling-up to the air-lift bioreactor decreased the biomass duplication time of PE cells up to 5.3 d post-inoculation (pi) and an average volumetric productivity of 1.6g/(dxL). Although slight browning was seen in the explants during the phase of 8-14 d pi, no losses in their viability and regenerative capability were observed. Cultured cells showed normal elongation in the transition from heart- to the torpedo-shape and finally to advanced developmental stages, with radicle emergence and whole plant generation. Agrobacterium-mediated transformation of cells was efficiently incorporated after this multiplication process by use of conventional procedures in dishes, allowing the generation of transgenic plantlets confirmed by PCR. PMID:18984020

  3. The Effects of Exogenous Auxins on Endogenous Indole-3-Acetic Acid Metabolism (The Implications for Carrot Somatic Embryogenesis).

    PubMed Central

    Ribnicky, D. M.; Ilic, N.; Cohen, J. D.; Cooke, T. J.

    1996-01-01

    The effect of auxin application on auxin metabolism was investigated in excised hypocotyl cultures of carrot (Daucus carota). Concentrations of both free and conjugated indole-3-acetic acid (IAA), [2H4]IAA, 2,4-dichlorophenoxyacetic acid, and naphthaleneacetic acid (NAA) were measured by mass spectroscopy using stable-isotope-labeled internal standards. [13C1]NAA was synthesized for this purpose, thus extending the range of auxins that can be assayed by stable-isotope techniques. 2,4-Dichlorophenoxyacetic acid promoted callus proliferation of the excised hypocotyls, accumulated as the free form in large quantities, and had minor effects on endogenous IAA concentrations. NAA promoted callus proliferation and the resulting callus became organogenic, producing both roots and shoots. NAA was found mostly in the conjugated form and had minor effects on endogenous IAA concentrations. [2H4]IAA had no visible effect on the growth pattern of cultured hypocotyls, possibly because it was rapidly metabolized to form inactive conjugates or possibly because it mediated a decrease in endogenous IAA concentrations by an apparent feedback mechanism. The presence of exogenous auxins did not affect tryptophan labeling of either the endogenous tryptophan or IAA pools. This suggested that exogenous auxins did not alter the IAA biosynthetic pathway, but that synthetic auxins did appear to be necessary to induce callus proliferation, which was essential for excised hypocotyls to gain the competence to form somatic embryos. PMID:12226408

  4. Enhanced induction of microspore embryogenesis after n-butanol treatment in wheat (Triticum aestivum L.) anther culture.

    PubMed

    Soriano, M; Cistué, L; Castillo, A M

    2008-05-01

    The aim of this study was the improvement of embryo production in wheat anther culture. Three butanol alcohols, n-butanol, sec-butanol and tert-butanol, were evaluated for their effect on microspore embryogenesis in two spring cultivars of wheat, Pavon and Caramba. Application of n-butanol, at 0.1 and 0.2% (v/v) in the induction media for 5 h, highly improved embryo production in both cultivars. Sec- and tert-butanol performed similarly to control plates. Regeneration ability was unaffected by any butyl-alcohol treatment. As a consequence of the higher embryo production after n-butanol treatment, the number of green regenerated plants increased up to five times in cultivar Pavon and up to three times in cultivar Caramba. The percentage of green plants was improved or unaffected by the treatment. Doubled haploid plant production was between 2 and 4 times higher after n-butanol treatment than in control plates. Therefore, n-butanol was successfully applied in the production of wheat doubled haploids. This primary alcohol is known as an activator of phospholipase D and has been previously reported to disrupt cortical microtubules and detach them from the plasma membrane in plants. Its effects on androgenetic induction could confirm the importance of microtubule regulation in plant cell fate, specifically in microspore development. A possible implication of phospholipase D is discussed. PMID:18214490

  5. Endogenous target mimics down-regulate miR160 mediation of ARF10, -16, and -17 cleavage during somatic embryogenesis in Dimocarpus longan Lour.

    PubMed

    Lin, Yuling; Lai, Zhongxiong; Tian, Qilin; Lin, Lixia; Lai, Ruilian; Yang, Manman; Zhang, Dongmin; Chen, Yukun; Zhang, Zihao

    2015-01-01

    MicroRNA160 plays a critical role in plant development by negatively regulating the auxin response factors ARF10, -16, and -17. However, the ways in which miR160 expression is regulated at the transcriptional level, and how miR160 interacts with its targets during plant embryo development, remain unknown. Here, we studied the regulatory relationships among endogenous target mimics (eTMs), and miR160 and its targets, and their involvement in hormone signaling and somatic embryogenesis (SE) in Dimocarpus longan. We identified miR160 family members and isolated the miR160 precursor, primary transcript, and promoter. The promoter contained cis-acting elements responsive to stimuli such as light, abscisic acid, salicylic acid (SA) and heat stress. The pri-miR160 was down-regulated in response to SA but up-regulated by gibberellic acid, ethylene, and methyl jasmonate treatment, suggesting that pri-miR160 was associated with hormone transduction. Dlo-miR160a, -a(∗) and -d(∗) reached expression peaks in torpedo-shaped embryos, globular embryos and cotyledonary embryos, respectively, but were barely detectable in friable-embryogenic callus. This suggests that they have expression-related and functional diversity, especially during the middle and later developmental stages of SE. Four potential eTMs for miR160 were identified. Two of them, glucan endo-1,3-beta- glucosidase-like protein 2-like and calpain-type cysteine protease DEK1, were confirmed to control the corresponding dlo-miR160a(∗) expression level. This suggests that they may function to abolish the binding between dlo-miR160a(∗) and its targets. These two eTMs also participated in 2,4-D and ABA signal transduction. DlARF10, -16, and -17 targeting by dlo-miR160a was confirmed; their expression levels were higher in friable-embryogenic callus and incomplete compact pro-embryogenic cultures and responded to 2,4-D, suggesting they may play a major role in the early stages of longan SE dependent on 2,4-D. The eTMs, miR160, and ARF10, -16, and -17 exhibited tissue specificity in 'Sijimi' longan vegetative and reproductive organs, but were not significant negatively correlated. These results provide insights into the possible role of the eTM-miR160-ARF10-16-17 pathway in longan somatic embryo development. PMID:26594219

  6. Endogenous target mimics down-regulate miR160 mediation of ARF10, -16, and -17 cleavage during somatic embryogenesis in Dimocarpus longan Lour

    PubMed Central

    Lin, Yuling; Lai, Zhongxiong; Tian, Qilin; Lin, Lixia; Lai, Ruilian; Yang, Manman; Zhang, Dongmin; Chen, Yukun; Zhang, Zihao

    2015-01-01

    MicroRNA160 plays a critical role in plant development by negatively regulating the auxin response factors ARF10, -16, and -17. However, the ways in which miR160 expression is regulated at the transcriptional level, and how miR160 interacts with its targets during plant embryo development, remain unknown. Here, we studied the regulatory relationships among endogenous target mimics (eTMs), and miR160 and its targets, and their involvement in hormone signaling and somatic embryogenesis (SE) in Dimocarpus longan. We identified miR160 family members and isolated the miR160 precursor, primary transcript, and promoter. The promoter contained cis-acting elements responsive to stimuli such as light, abscisic acid, salicylic acid (SA) and heat stress. The pri-miR160 was down-regulated in response to SA but up-regulated by gibberellic acid, ethylene, and methyl jasmonate treatment, suggesting that pri-miR160 was associated with hormone transduction. Dlo-miR160a, -a∗ and -d∗ reached expression peaks in torpedo-shaped embryos, globular embryos and cotyledonary embryos, respectively, but were barely detectable in friable-embryogenic callus. This suggests that they have expression-related and functional diversity, especially during the middle and later developmental stages of SE. Four potential eTMs for miR160 were identified. Two of them, glucan endo-1,3-beta- glucosidase-like protein 2-like and calpain-type cysteine protease DEK1, were confirmed to control the corresponding dlo-miR160a∗ expression level. This suggests that they may function to abolish the binding between dlo-miR160a∗ and its targets. These two eTMs also participated in 2,4-D and ABA signal transduction. DlARF10, -16, and -17 targeting by dlo-miR160a was confirmed; their expression levels were higher in friable-embryogenic callus and incomplete compact pro-embryogenic cultures and responded to 2,4-D, suggesting they may play a major role in the early stages of longan SE dependent on 2,4-D. The eTMs, miR160, and ARF10, -16, and -17 exhibited tissue specificity in ‘Sijimi’ longan vegetative and reproductive organs, but were not significant negatively correlated. These results provide insights into the possible role of the eTM-miR160-ARF10-16-17 pathway in longan somatic embryo development. PMID:26594219

  7. Evaluation of suitable reference genes for normalization of microRNA expression by real-time reverse transcription PCR analysis during longan somatic embryogenesis.

    PubMed

    Lin, Yu Ling; Lai, Zhong Xiong

    2013-05-01

    Accurate profiling of microRNAs (miRNAs) is an essential step for understanding both developmental and physiological functions of miRNAs. Real-time quantitative PCR (qPCR) is being widely used in miRNA expression studies, but choosing a suitable reference gene is a crucial factor for correct analysis of results. To date, there has been no systematic evaluation of qPCR reference genes for the study of miRNAs during somatic embryogenesis (SE) in the longan tree (Dimocarpus longan). Here, the most stably expressed miRNAs in synchronized longan tree embryogenic cultures at different developmental stages were determined using the geNorm and NormFinder algorithms. Validation qPCR experiments were performed for 24 miRNAs together with a snRNA (U6 snRNA), a rRNA (5S rRNA), and three housekeeping genes. It was found that small RNAs had better expression stability than protein-coding genes, and dlo-miR24 was identified as the most reliable reference gene, followed by dlo-miR168a*, dlo-miR2089*-1 and 5S rRNA. dlo-miR24 was recommended as a normalizer if only a single reference gene was to be used, while the combination of dlo-miR156c, dlo-2089*-1 and 5S rRNA was preferred to normalize miRNA expression data during longan SE. PMID:23454294

  8. Induction of Embryogenesis in Brassica Napus Microspores Produces a Callosic Subintinal Layer and Abnormal Cell Walls with Altered Levels of Callose and Cellulose

    PubMed Central

    Parra-Vega, Verónica; Corral-Martínez, Patricia; Rivas-Sendra, Alba; Seguí-Simarro, Jose M.

    2015-01-01

    The induction of microspore embryogenesis produces dramatic changes in different aspects of the cell physiology and structure. Changes at the cell wall level are among the most intriguing and poorly understood. In this work, we used high pressure freezing and freeze substitution, immunolocalization, confocal, and electron microscopy to analyze the structure and composition of the first cell walls formed during conventional Brassica napus microspore embryogenesis, and in cultures treated to alter the intracellular Ca2+ levels. Our results revealed that one of the first signs of embryogenic commitment is the formation of a callose-rich, cellulose-deficient layer beneath the intine (the subintinal layer), and of irregular, incomplete cell walls. In these events, Ca2+ may have a role. We propose that abnormal cell walls are due to a massive callose synthesis and deposition of excreted cytoplasmic material, and the parallel inhibition of cellulose synthesis. These features were absent in pollen-like structures and in microspore-derived embryos, few days after the end of the heat shock, where abnormal cell walls were no longer produced. Together, our results provide an explanation to a series of relevant aspects of microspore embryogenesis including the role of Ca2+ and the occurrence of abnormal cell walls. In addition, our discovery may be the explanation to why nuclear fusions take place during microspore embryogenesis. PMID:26635844

  9. MicroRNA390-Directed TAS3 Cleavage Leads to the Production of tasiRNA-ARF3/4 During Somatic Embryogenesis in Dimocarpus longan Lour

    PubMed Central

    Lin, Yuling; Lin, Lixia; Lai, Ruilian; Liu, Weihua; Chen, Yukun; Zhang, Zihao; XuHan, Xu; Lai, Zhongxiong

    2015-01-01

    Trans-acting short-interfering RNAs (tasiRNAs) originate from TAS3 families through microRNA (miRNA) 390-guided cleavage of primary transcripts and target auxin response factors (ARF3/-4), which are involved in the normal development of lateral roots and flowers in plants. However, their roles in embryo development are still unclear. Here, the pathway miR390-TAS3-ARF3/-4 was identified systematically for the first time during somatic embryo development in Dimocarpus longan. We identified the miR390 primary transcript and promoter. The promoter contained cis-acting elements responsive to stimuli such as light, salicylic acid, anaerobic induction, fungal elicitor, circadian control, and heat stress. The longan TAS3 transcript, containing two miR390-binding sites, was isolated; the miR390- guided cleavage site located near the 3′ end of the TAS3 transcript was verified. Eight TAS3-tasiRNAs with the 21-nucleotides phase were found among longan small RNA data, further confirming that miR390-directed TAS3 cleavage leads to the production of tasiRNA in longan. Among them, TAS3_5′D5+ and 5′D6+ tasiRNAs were highly abundant, and verified to target ARF3 and -4, implying that miR390-guided TAS3 cleavage with 21-nucleotides phase leading to the production of tasiRNA-ARF is conserved in plants. Pri-miR390 was highly expressed in friable-embryogenic callus (EC), and less expressed in incomplete compact pro-embryogenic cultures, while miR390 showed its lowest expression in EC and highest expression in torpedo-shaped embryos (TEs). DlTAS3 and DlARF4 both exhibited their lowest expressions in EC, and reached their peaks in the globular embryos stage, which were mainly inversely proportional to the expression of miR390, especially at the globular embryos to cotyledonary embryos (CEs) stages. While DlARF3 showed little variation from the EC to TEs stages, and exhibited its lowest expression in the CEs stage. There was a general lack of correlation between the expressions of DlARF3 and miR390. In addition, pri-miR390, DlTAS3, DlARF3 and -4 were up-regulated by 2,4-D in a concentration-dependent manner. They were also preferentially expressed in roots, pulp, and seeds of ‘Sijimi’ longan, implying their extended roles in the development of longan roots and fruit. This study provided insights into a possible role of miR390-tasiRNAs-ARF in plant somatic embryo development. PMID:26734029

  10. MicroRNA390-Directed TAS3 Cleavage Leads to the Production of tasiRNA-ARF3/4 During Somatic Embryogenesis in Dimocarpus longan Lour.

    PubMed

    Lin, Yuling; Lin, Lixia; Lai, Ruilian; Liu, Weihua; Chen, Yukun; Zhang, Zihao; XuHan, Xu; Lai, Zhongxiong

    2015-01-01

    Trans-acting short-interfering RNAs (tasiRNAs) originate from TAS3 families through microRNA (miRNA) 390-guided cleavage of primary transcripts and target auxin response factors (ARF3/-4), which are involved in the normal development of lateral roots and flowers in plants. However, their roles in embryo development are still unclear. Here, the pathway miR390-TAS3-ARF3/-4 was identified systematically for the first time during somatic embryo development in Dimocarpus longan. We identified the miR390 primary transcript and promoter. The promoter contained cis-acting elements responsive to stimuli such as light, salicylic acid, anaerobic induction, fungal elicitor, circadian control, and heat stress. The longan TAS3 transcript, containing two miR390-binding sites, was isolated; the miR390- guided cleavage site located near the 3' end of the TAS3 transcript was verified. Eight TAS3-tasiRNAs with the 21-nucleotides phase were found among longan small RNA data, further confirming that miR390-directed TAS3 cleavage leads to the production of tasiRNA in longan. Among them, TAS3_5'D5+ and 5'D6+ tasiRNAs were highly abundant, and verified to target ARF3 and -4, implying that miR390-guided TAS3 cleavage with 21-nucleotides phase leading to the production of tasiRNA-ARF is conserved in plants. Pri-miR390 was highly expressed in friable-embryogenic callus (EC), and less expressed in incomplete compact pro-embryogenic cultures, while miR390 showed its lowest expression in EC and highest expression in torpedo-shaped embryos (TEs). DlTAS3 and DlARF4 both exhibited their lowest expressions in EC, and reached their peaks in the globular embryos stage, which were mainly inversely proportional to the expression of miR390, especially at the globular embryos to cotyledonary embryos (CEs) stages. While DlARF3 showed little variation from the EC to TEs stages, and exhibited its lowest expression in the CEs stage. There was a general lack of correlation between the expressions of DlARF3 and miR390. In addition, pri-miR390, DlTAS3, DlARF3 and -4 were up-regulated by 2,4-D in a concentration-dependent manner. They were also preferentially expressed in roots, pulp, and seeds of 'Sijimi' longan, implying their extended roles in the development of longan roots and fruit. This study provided insights into a possible role of miR390-tasiRNAs-ARF in plant somatic embryo development. PMID:26734029

  11. Protocols for Callus and Somatic Embryo Initiation for Hibiscus sabdariffa L. (Malvaceae): Influence of Explant Type, Sugar, and Plant Growth Regulators

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A significant work on callus induction and somatic embryogenesis was realized for Hibiscus sabdariffa. Two genotypes (Hibiscus sabdariffa and Hibiscus sabdariffa var. altissima) two sugars (sucrose and glucose) and three concentrations (1 %, 2%, 3%) of each sugar, 3 explant types (root, hypocotyl, c...

  12. Recent Advances on Genetic and Physiological Bases of In Vitro Somatic Embryo Formation.

    PubMed

    Altamura, Maria Maddalena; Della Rovere, Federica; Fattorini, Laura; D'Angeli, Simone; Falasca, Giuseppina

    2016-01-01

    Somatic embryogenesis involves a broad repertoire of genes, and complex expression patterns controlled by a concerted gene regulatory network. The present work describes this regulatory network focusing on the main aspects involved, with the aim of providing a deeper insight into understanding the total reprogramming of cells into a new organism through a somatic way. To the aim, the chromatin remodeling necessary to totipotent stem cell establishment is described, as the activity of numerous transcription factors necessary to cellular totipotency reprogramming. The eliciting effects of various plant growth regulators on the induction of somatic embryogenesis is also described and put in relation with the activity of specific transcription factors. The role of programmed cell death in the process, and the related function of specific hemoglobins as anti-stress and anti-death compounds is also described. The tools for biotechnology coming from this information is highlighted in the concluding remarks. PMID:26619858

  13. Endogenous Gibberellin-Like Substances in Somatic Embryos of Grape (Vitis vinifera × Vitis rupestris) in Relation to Embryogenesis and the Chilling Requirement for Subsequent Development of Mature Embryos 1

    PubMed Central

    Takeno, Kiyotoshi; Koshioka, Masaji; Pharis, Richard P.; Rajasekaran, K.; Mullins, Michael G.

    1983-01-01

    Endogenous gibberellin (GA)-like substances were examined in suspension cultures of somatic embryos of a hybrid grape (Vitis vinifera × Vitis rupestris) during embryogenesis, and in mature embryos chilled at 4°C, and subsequently incubated at 26°C with and without abscisic acid (ABA). The extract was separated into a nonpolar fraction (would contain GA-precursors); a fraction that would contain free GAs; and a highly H2O-soluble fraction (would contain GA glucosyl conjugates and very polar free GAs). Quantitation after SiO2 partition chromatography was accomplished by microdrop and immersion dwarf rice bioassays. As embryogenesis developed, the free and highly H2O-soluble GA-like substances, expressed on a dry weight basis, decreased (however, they increased on a per embryo basis). Chilling at 4°C for 1 week greatly increased activity of free GA-like substances (per g dry weight and per embryo), it then declined over the next three weeks of chilling. Activity (per g dry weight and per embryo) in the H2O-soluble fraction declined throughout chilling. Activity in the GA-precursor fraction, however, increased steadily with chilling (per g dry weight and per embryo). Incubation at 26°C after chilling enhanced activity in the free GA and H2O-soluble fractions (per g dry weight and per embryo), but activity in the GA-precursor fraction dropped dramatically. Incubation at 26°C with (±) ABA after chilling prevented germination and maintained high activity for GA precursors and less polar free GAs and low activity in the polar free GA and H2O-soluble fractions. Kaurene and kaurenoic acid were characterized in the GA-precursor fraction of chilled embryos by gas-liquid chromatography-mass spectrometry (GLC-MS). The existence of GA4 and GA9 in ABA-treated, chilled embryos was also confirmed by GLC-MS. PMID:16663304

  14. Nonimmunogenic radiation-induced lymphoma: immunity induction by a somatic cell hybrid

    SciTech Connect

    Yefenof, E.; Goldapfel, M.; Ber, R.

    1982-05-01

    The cell line designated PIR-2 is a nonimmunogenic X-ray-induced thymoma of C57BL/6 origin that is unable to induce antitumor immunity in syngeneic lymphocytes in vitro and in mice in vivo. Fusion of PIR-2 with an allogeneic universal fuser A9HT (clone 3c) resulted in the establishment of a somatic cell hybrid designated A9/PIR. C57BL/6 lymphocytes sensitized in vitro with A9/PIR could lyse parental PIR-2 cells, as well as other syngeneic tumors. However, immunization of mice with the hybrid significantly enhanced PIR-2 tumor takes while it partially protected the animals against a challenge with unrelated syngeneic tumors. The results imply that somatic cell hybridization can increase the immunogenicity of an otherwise nonimmunogenic tumor. However, in view of the enhancing effects of hybrid preimmunization on parental tumor cell growth, the possible application of this approach for immunotherapy is questionable.

  15. Bioreactors for Plant Embryogenesis and Beyond.

    PubMed

    Fei, Liwen; Weathers, Pamela

    2016-01-01

    A variety of different bioreactors have been developed for use in initiating and cultivating somatic embryos. The various designs for embryogenesis and culture are critically evaluated here. Bioreactor optimization and operation methods are also described along with recommendations for use based on desired outcome. PMID:26619865

  16. Screening of medicinal plants for induction of somatic segregation activity in Aspergillus nidulans.

    PubMed

    Ramos Ruiz, A; De la Torre, R A; Alonso, N; Villaescusa, A; Betancourt, J; Vizoso, A

    1996-07-01

    Knowledge about mutagenic properties of plants commonly used in traditional medicine is limited. A screening for genotoxic activity was carried out in aqueous or alcoholic extracts prepared from 13 medicinal plants widely used as folk medicine in Cuba: Lepidium virginicum L. (Brassicaceae): Plantago major L. and Plantago lanceolata L. (Plantaginaceae); Ortosiphon aristatus Blume, Mentha x piperita L., Melissa officinalis L. and Plectranthus amboinicus (Lour.) Spreng. (Lamiaceae); Cymbopogon citratus (DC.) Stapf (Poaceae); Passiflora incarnata L. (Passifloraceae); Zingiber officinale Roscoe (Zingiberaceae); Piper auritum HBK. (Piperaceae); Schinus terebinthifolius Raddi (Anacardeaceae) and Momordica charantia L. (Cucurbitaceae). A plate incorporation assay with Aspergillus nidulans was employed, allowing detection of somatic segregation as a result of mitotic crossing-over, chromosome malsegregation or clastogenic effects. Aspergillus nidulans D-30, a well-marked strain carrying four recessive mutations for conidial color in heterozygosity, which permitted the direct visual detection of segregants, was used throughout this study. As a result, only in the aqueous extract of one of the plants screened (Momordica charantia) a statistical significant increase in the frequency of segregant sectors per colony was observed, and consequently, a genotoxic effect is postulated. PMID:8771452

  17. Developmental pattern formation of somatic embryos induced in cell suspension cultures of cowpea [Vigna unguiculata (L.) Walp].

    PubMed

    Ramakrishnan, K; Gnanam, R; Sivakumar, P; Manickam, A

    2005-11-01

    The sequence of events in the functional body pattern formation during the somatic embryo development in cowpea suspensions is described under three heads. Early stages of somatic embryogenesis were characterized by both periclinal and anti-clinal cell divisions. Differentiation of the protoderm cell layer by periclinal divisions marked the commencement of somatic embryogenesis. The most critical events appear to be the formation of apical meristems, establishment of apical-basal patterns of symmetry, and cellular organization in oblong-stage somatic embryo for the transition to torpedo and cotyledonary-stage somatic embryos. Two different stages of mature embryos showing distinct morphology, classified based on the number of cotyledons and their ability to convert into plantlets, were visualized. Repeated mitotic divisions of the sub-epidermal cell layers marked the induction of pro-embryogenic mass (PEM) in the embryogenic calli. The first division plane was periclinally-oriented, the second anti-clinally-oriented, and the subsequent division planes appeared in any direction, leading to clusters of pro-embryogenic clumps. Differentiation of the protoderm layer marks the beginning of the structural differentiation in globular stage. Incipient pro-cambium formation is the first sign of somatic embryo transition. Axial elongation of inner isodiametric cells of the globular somatic embryo followed by the change in the growth axis of the pro-cambium is an important event in oblong-stage somatic embryo. Vacuolation in the ground meristem of torpedo-stage embryo begins the process of histodifferentiation. Three major embryonic tissue systems; shoot apical meristem, root apical meristem, and the differentiation of pro-cambial strands, are visible in torpedo-stage somatic embryo. Monocotyledonary-stage somatic embryo induced both the shoot apical meristem and two leaf primordia compared to the ansiocotyledonary somatic embryo. PMID:15959730

  18. Induction of pluripotent stem cells from autopsy donor-derived somatic cells.

    PubMed

    Hjelm, Brooke E; Rosenberg, Jon B; Szelinger, Szabolcs; Sue, Lucia I; Beach, Thomas G; Huentelman, Matthew J; Craig, David W

    2011-09-20

    Human induced pluripotent stem cells (iPSCs) have become an intriguing approach for neurological disease modeling, because neural lineage-specific cell types that retain the donors' complex genetics can be established in vitro. The statistical power of these iPSC-based models, however, is dependent on accurate diagnoses of the somatic cell donors; unfortunately, many neurodegenerative diseases are commonly misdiagnosed in live human subjects. Postmortem histopathological examination of a donor's brain, combined with premortem clinical criteria, is often the most robust approach to correctly classify an individual as a disease-specific case or unaffected control. In this study, we describe iPSCs generated from a skin biopsy collected postmortem during the rapid autopsy of a 75-year-old male, whole body donor, defined as an unaffected neurological control by both clinical and histopathological criteria. These iPSCs were established in a feeder-free system by lentiviral transduction of the Yamanaka factors, Oct3/4, Sox2, Klf4, and c-Myc. Selected iPSC clones expressed both nuclear and surface antigens recognized as pluripotency markers of human embryonic stem cells (hESCs) and were able to differentiate in vitro into neurons and glia. Statistical analysis also demonstrated that fibroblast proliferation was significantly affected by biopsy site, but not donor age (within an elderly cohort). These results provide evidence that autopsy donor-derived fibroblasts can be successfully reprogrammed into iPSCs, and may provide an advantageous approach for generating iPSC-based neurological disease models. PMID:21839145

  19. Technological Overview of iPS Induction from Human Adult Somatic Cells

    PubMed Central

    Bayart, Emilie; Cohen-Haguenauer, Odile

    2013-01-01

    The unlimited proliferation capacity of embryonic stem cells (ESCs) combined with their pluripotent differentiation potential in various lineages raised great interest in both the scientific community and the public at large with hope for future prospects of regenerative medicine. However, since ESCs are derived from human embryos, their use is associated with significant ethical issues preventing broad studies and therapeutic applications. To get around this bottleneck, Takahashi and Yamanaka have recently achieved the conversion of adult somatic cells into ES-like cells via the forced expression of four transcription factors: Oct3/4, Sox2, Klf4 and c-Myc. This first demonstration attracted public attention and opened a new field of stem cells research with both cognitive – such as disease modeling - and therapeutic prospects. This pioneer work just received the 2012 Nobel Prize in Physiology or Medicine. Many methods have been reported since 2006, for the generation of induced pluripotent stem (iPS) cells. Most strategies currently under use are based on gene delivery via gamma-retroviral or lentiviral vectors; some experiments have also been successful using plasmids or transposons-based systems and few with adenovirus. However, most experiments involve integration in the host cell genome with an identified risk for insertional mutagenesis and oncogenic transformation. To circumvent such risks which are deemed incompatible with therapeutic prospects, significant progress has been made with transgene-free reprogramming methods based on e.g.: sendaï virus or direct mRNA or protein delivery to achieve conversion of adult cells into iPS. In this review we aim to cover current knowledge relating to both delivery systems and combinations of inducing factors including chemicals which are used to generate human iPS cells. Finally, genetic instability resulting from the reprogramming process is also being considered as a safety bottleneck for future clinical translation and stem cell-therapy prospects based on iPS. PMID:23320476

  20. Vitamin C deficiency improves somatic embryo development through distinct gene regulatory networks in Arabidopsis

    PubMed Central

    Becker, Michael G.; Chan, Ainsley; Mao, Xingyu; Girard, Ian J.; Lee, Samantha; Elhiti, Mohamed; Stasolla, Claudio; Belmonte, Mark F.

    2014-01-01

    Changes in the endogenous ascorbate redox status through genetic manipulation of cellular ascorbate levels were shown to accelerate cell proliferation during the induction phase and improve maturation of somatic embryos in Arabidopsis. Mutants defective in ascorbate biosynthesis such as vtc2-5 contained ~70 % less cellular ascorbate compared with their wild-type (WT; Columbia-0) counterparts. Depletion of cellular ascorbate accelerated cell division processes and cellular reorganization and improved the number and quality of mature somatic embryos grown in culture by 6-fold compared with WT tissues. To gain insight into the molecular mechanisms underlying somatic embryogenesis (SE), we profiled dynamic changes in the transcriptome and analysed dominant patterns of gene activity in the WT and vtc2-5 lines across the somatic embryo culturing process. Our results provide insight into the gene regulatory networks controlling SE in Arabidopsis based on the association of transcription factors with DNA sequence motifs enriched in biological processes of large co-expressed gene sets. These data provide the first detailed account of temporal changes in the somatic embryo transcriptome starting with the zygotic embryo, through tissue dedifferentiation, and ending with the mature somatic embryo, and impart insight into possible mechanisms for the improved culture of somatic embryos in the vtc2-5 mutant line. PMID:25151615

  1. Vitamin C deficiency improves somatic embryo development through distinct gene regulatory networks in Arabidopsis.

    PubMed

    Becker, Michael G; Chan, Ainsley; Mao, Xingyu; Girard, Ian J; Lee, Samantha; Elhiti, Mohamed; Stasolla, Claudio; Belmonte, Mark F

    2014-11-01

    Changes in the endogenous ascorbate redox status through genetic manipulation of cellular ascorbate levels were shown to accelerate cell proliferation during the induction phase and improve maturation of somatic embryos in Arabidopsis. Mutants defective in ascorbate biosynthesis such as vtc2-5 contained ~70 % less cellular ascorbate compared with their wild-type (WT; Columbia-0) counterparts. Depletion of cellular ascorbate accelerated cell division processes and cellular reorganization and improved the number and quality of mature somatic embryos grown in culture by 6-fold compared with WT tissues. To gain insight into the molecular mechanisms underlying somatic embryogenesis (SE), we profiled dynamic changes in the transcriptome and analysed dominant patterns of gene activity in the WT and vtc2-5 lines across the somatic embryo culturing process. Our results provide insight into the gene regulatory networks controlling SE in Arabidopsis based on the association of transcription factors with DNA sequence motifs enriched in biological processes of large co-expressed gene sets. These data provide the first detailed account of temporal changes in the somatic embryo transcriptome starting with the zygotic embryo, through tissue dedifferentiation, and ending with the mature somatic embryo, and impart insight into possible mechanisms for the improved culture of somatic embryos in the vtc2-5 mutant line. PMID:25151615

  2. Dying with Style: Death Decision in Plant Embryogenesis.

    PubMed

    Huang, Shuanglong; Mira, Mohamed M; Stasolla, Claudio

    2016-01-01

    Embryogenesis is a fascinating event during the plant life cycle encompassing several steps whereby the zygote develops into a fully developed embryo which, in angiosperms, is composed of an axis separating the apical meristems, and two cotyledons. Recapitulation of embryogenesis can also occur in vitro through somatic embryogenesis, where somatic cells are induced to form embryos, and androgenesis, in which embryos originate from immature male gametophytes. Besides cell division and differentiation, embryo patterning in vivo and in vitro requires the dismantling and selective elimination of cells and tissues via programmed cell death (PCD). While the manifestation of the death program has long been acknowledged in vivo, especially in relation to the elimination of the suspensor during the late phases of embryo development, PCD during in vitro embryogenesis has only been described in more recent years. Independent studies using the gymnosperm Norway spruce and the angiosperm maize have shown that the death program is crucial for the proper formation and further development of immature somatic embryos. This chapter summarizes the recent advances in the field of PCD during embryogenesis and proposes novel regulatory mechanisms activating the death program in plants. PMID:26619860

  3. Enhancement of American chestnut somatic seedling production.

    PubMed

    Andrade, G M; Merkle, S A

    2005-08-01

    Somatic embryogenesis holds promise for mass propagation of American chestnut trees bred or genetically engineered for resistance to chestnut blight. However, low germination frequency of chestnut somatic embryos has limited somatic seedling production for this forest tree. We tested the effects of culture regime (semi-solid versus liquid), cold treatment, AC and somatic embryo morphology (i.e., cotyledon number) on germination and conversion of the somatic embryos. Cold treatment for 12 weeks was critical for conversion of chestnut somatic embryos to somatic seedlings, raising conversion frequencies for one line to 47%, compared to 7% with no cold treatment. AC improved germination and conversion frequency for one line to 77% and 59%, respectively, and kept roots from darkening. For two lines that produced embryos with one, two or three-plus cotyledons, cotyledon number did not affect germination or conversion frequency. We also established embryogenic American chestnut suspension cultures and adapted a fractionation/plating system that allowed us to produce populations of relatively synchronous somatic embryos for multiple lines. Embryos derived from suspension cultures of two lines tested had higher conversion frequencies (46% and 48%) than those from cultures maintained on semi-solid medium (7% and 30%). The improvements in manipulation of American chestnut embryogenic cultures described in this study have allowed over a 100-fold increase in somatic seedling production efficiency over what we reported previously and thus constitute a substantial advance toward the application of somatic embryogenesis for mass clonal propagation of the tree. PMID:15789206

  4. Induction of pluripotency in human somatic cells via a transient state resembling primitive streak-like mesendoderm.

    PubMed

    Takahashi, Kazutoshi; Tanabe, Koji; Ohnuki, Mari; Narita, Megumi; Sasaki, Aki; Yamamoto, Masamichi; Nakamura, Michiko; Sutou, Kenta; Osafune, Kenji; Yamanaka, Shinya

    2014-01-01

    During mammalian embryonic development, the primitive streak initiates the differentiation of pluripotent epiblast cells into germ layers. Pluripotency can be reacquired in committed somatic cells using a combination of a handful of transcription factors, such as OCT3/4, SOX2, KLF4 and c-MYC (hereafter referred to as OSKM), albeit with low efficiency. Here we show that during OSKM-induced reprogramming towards pluripotency in human cells, intermediate cells transiently show gene expression profiles resembling mesendoderm, which is a major component of the primitive streak. Based on these findings, we discover that forkhead box H1 (FOXH1), a transcription factor required for anterior primitive streak specification during early development, significantly enhances the reprogramming efficiency of human fibroblasts by promoting their maturation, including mesenchymal to epithelial transition and the activation of late pluripotency markers. These results demonstrate that during the reprogramming process, human somatic cells go through a transient state that resembles mesendoderm. PMID:24759836

  5. A Comparison of In Vitro and In Vivo Asexual Embryogenesis.

    PubMed

    Hand, Melanie L; de Vries, Sacco; Koltunow, Anna M G

    2016-01-01

    In plants, embryogenesis generally occurs through the sexual process of double fertilization, which involves a haploid sperm cell fusing with a haploid egg cell to ultimately give rise to a diploid embryo. Embryogenesis can also occur asexually in the absence of fertilization, both in vitro and in vivo. Somatic or gametic cells are able to differentiate into embryos in vitro following the application of plant growth regulators or stress treatments. Asexual embryogenesis also occurs naturally in some plant species in vivo, from either ovule cells as part of a process defined as apomixis, or from somatic leaf tissue in other species. In both in vitro and in vivo asexual embryogenesis, the embryo precursor cells must attain an embryogenic fate without the act of fertilization. This review compares the processes of in vitro and in vivo asexual embryogenesis including what is known regarding the genetic and epigenetic regulation of each process, and considers how the precursor cells are able to change fate and adopt an embryogenic pathway. PMID:26619856

  6. Comparison of sister chromatid exchange induction in murine germinal and somatic cells by gamma radiation exposure in vivo

    SciTech Connect

    Morales-Ramirez, P.; Mendiola-Cruz, M.T.; Vallarino-Kelly, T.; Rodriguez-Reyes, R.

    1994-12-31

    Sister chromatid exchange (SCE) induction by gamma rays was determined in speratogonia irradiated before or after BrdU incorporation. Furthermore, the comparison of responses obtained in spermatogonia, bone marrow and salivary gland cells was carried out in the cells irradiated after BrdU incorporation, a condition which permits a higher SCE induction. Results indicate that gamma ray exposure of spermatogonia could induce a significant increase in SCE frequency with doses as low as 0.27 Gy, either before or after BrdU incorporation. However, the increase caused by radiation exposure after BrdU incorporation in spermatogonia was nearly three times lower than that obtained in both bone marrow and salivary gland cells. These data suggest that spermatogonia are either more efficient in repairing the gamma ray-induced lesions involved in SCE production or that these cells are less prone to the induction of such lesions. 53 refs., 2 figs., 3 tabs.

  7. THE REGENERATION OF BLACK GRAMA PLANTS VIA SOMATIC EMBRYOGENESIS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Seeds of Bouteloua eropida, (Torr.) Torr. were surface disinfested and germinated on a carbon mineral rich medium. Callus was initiated from embryonic shoots excised from the roots on auxin supplemented medium. After callus multiplication, embryos were induced from callus. They developed into nor...

  8. Transcriptional identification and characterization of differentially expressed genes associated with embryogenesis in radish (Raphanus sativus L.)

    PubMed Central

    Zhai, Lulu; Xu, Liang; Wang, Yan; Zhu, Xianwen; Feng, Haiyang; Li, Chao; Luo, Xiaobo; Everlyne, Muleke M.; Liu, Liwang

    2016-01-01

    Embryogenesis is an important component in the life cycle of most plant species. Due to the difficulty in embryo isolation, the global gene expression involved in plant embryogenesis, especially the early events following fertilization are largely unknown in radish. In this study, three cDNA libraries from ovules of radish before and after fertilization were sequenced using the Digital Gene Expression (DGE) tag profiling strategy. A total of 5,777 differentially expressed transcripts were detected based on pairwise comparison in the three libraries (0_DAP, 7_DAP and 15_DAP). Results from Gene Ontology (GO) and pathway enrichment analysis revealed that these differentially expressed genes (DEGs) were implicated in numerous life processes including embryo development and phytohormones biosynthesis. Notably, some genes encoding auxin response factor (ARF ), Leafy cotyledon1 (LEC1) and somatic embryogenesis receptor-like kinase (SERK ) known to be involved in radish embryogenesis were differentially expressed. The expression patterns of 30 genes including LEC1-2, AGL9, LRR, PKL and ARF8-1 were validated by qRT-PCR. Furthermore, the cooperation between miRNA and mRNA may play a pivotal role in the radish embryogenesis process. This is the first report on identification of DEGs profiles related to radish embryogenesis and seed development. These results could facilitate further dissection of the molecular mechanisms underlying embryogenesis and seed development in radish. PMID:26902837

  9. Transcriptional identification and characterization of differentially expressed genes associated with embryogenesis in radish (Raphanus sativus L.).

    PubMed

    Zhai, Lulu; Xu, Liang; Wang, Yan; Zhu, Xianwen; Feng, Haiyang; Li, Chao; Luo, Xiaobo; Everlyne, Muleke M; Liu, Liwang

    2016-01-01

    Embryogenesis is an important component in the life cycle of most plant species. Due to the difficulty in embryo isolation, the global gene expression involved in plant embryogenesis, especially the early events following fertilization are largely unknown in radish. In this study, three cDNA libraries from ovules of radish before and after fertilization were sequenced using the Digital Gene Expression (DGE) tag profiling strategy. A total of 5,777 differentially expressed transcripts were detected based on pairwise comparison in the three libraries (0_DAP, 7_DAP and 15_DAP). Results from Gene Ontology (GO) and pathway enrichment analysis revealed that these differentially expressed genes (DEGs) were implicated in numerous life processes including embryo development and phytohormones biosynthesis. Notably, some genes encoding auxin response factor (ARF ), Leafy cotyledon1 (LEC1) and somatic embryogenesis receptor-like kinase (SERK ) known to be involved in radish embryogenesis were differentially expressed. The expression patterns of 30 genes including LEC1-2, AGL9, LRR, PKL and ARF8-1 were validated by qRT-PCR. Furthermore, the cooperation between miRNA and mRNA may play a pivotal role in the radish embryogenesis process. This is the first report on identification of DEGs profiles related to radish embryogenesis and seed development. These results could facilitate further dissection of the molecular mechanisms underlying embryogenesis and seed development in radish. PMID:26902837

  10. Initiation of somatic embryos and regeneration of plants from primordial shoots of 10-year-old somatic white spruce and expression profiles of 11 genes followed during the tissue culture process.

    PubMed

    Klimaszewska, Krystyna; Overton, Catherine; Stewart, Don; Rutledge, Robert G

    2011-03-01

    Adult conifers are notoriously recalcitrant in vegetative propagation and micropropagation that would result in the regeneration of juvenile propagules through somatic embryogenesis (SE) has not been demonstrated to date. Because SE-derived material is more amenable in subsequent tissue culture experiments compared with seed-derived material, a multi-year study was conducted to investigate induction of SE from primordial shoot (PS) explants that were excised from shoot buds of somatic embryo-derived white spruce. The SE induction experiments were carried out first with greenhouse-grown and later with field-grown trees each year from 2002 (2-year-old) to 2010 (10-year-old). Of the four genotypes tested, 893-2 and 893-12 never responded, 893-1 responded up to year 4 and 893-6 consistently responded every year. In 2010, for the first time, three of the 17 893-6 clonal trees produced male strobili as well as SE from cultured PS explants. SE induction was associated with formation of a nodule on the surface of an elongated needle primordium or in callus. Early somatic embryos were detectable after about 3 weeks of culture. Of 11 genes whose expression profiles were followed during the PS cultures, CHAP3A, VP1, WOX2 and SAP2C were expressed exclusively in the early stages of SE, and could potentially be used as markers of embryogenecity. Mature somatic embryos and plants were produced from the explants of responding genotype. Implication of these results for future research on adult conifer recalcitrance in micropropagation is discussed. PMID:21136075

  11. Human somatic cells subjected to genetic induction with six germ line-related factors display meiotic germ cell-like features.

    PubMed

    Medrano, Jose V; Martínez-Arroyo, Ana M; Míguez, Jose M; Moreno, Inmaculada; Martínez, Sebastián; Quiñonero, Alicia; Díaz-Gimeno, Patricia; Marqués-Marí, Ana I; Pellicer, Antonio; Remohí, Jose; Simón, Carlos

    2016-01-01

    The in vitro derivation of human germ cells has attracted interest in the last years, but their direct conversion from human somatic cells has not yet been reported. Here we tested the ability of human male somatic cells to directly convert into a meiotic germ cell-like phenotype by inducing them with a combination of selected key germ cell developmental factors. We started with a pool of 12 candidates that were reduced to 6, demonstrating that ectopic expression of the germ line-related genes PRDM1, PRDM14, LIN28A, DAZL, VASA and SYCP3 induced direct conversion of somatic cells (hFSK (46, XY), and hMSC (46, XY)) into a germ cell-like phenotype in vitro. Induced germ cell-like cells showed a marked switch in their transcriptomic profile and expressed several post-meiotic germ line related markers, showed meiotic progression, evidence of epigenetic reprogramming, and approximately 1% were able to complete meiosis as demonstrated by their haploid status and the expression of several post-meiotic markers. Furthermore, xenotransplantation assays demonstrated that a subset of induced cells properly colonize the spermatogonial niche. Knowledge obtained from this work can be used to create in vitro models to study gamete-related diseases in humans. PMID:27112843

  12. Human somatic cells subjected to genetic induction with six germ line-related factors display meiotic germ cell-like features

    PubMed Central

    Medrano, Jose V.; Martínez-Arroyo, Ana M.; Míguez, Jose M.; Moreno, Inmaculada; Martínez, Sebastián; Quiñonero, Alicia; Díaz-Gimeno, Patricia; Marqués-Marí, Ana I.; Pellicer, Antonio; Remohí, Jose; Simón, Carlos

    2016-01-01

    The in vitro derivation of human germ cells has attracted interest in the last years, but their direct conversion from human somatic cells has not yet been reported. Here we tested the ability of human male somatic cells to directly convert into a meiotic germ cell-like phenotype by inducing them with a combination of selected key germ cell developmental factors. We started with a pool of 12 candidates that were reduced to 6, demonstrating that ectopic expression of the germ line-related genes PRDM1, PRDM14, LIN28A, DAZL, VASA and SYCP3 induced direct conversion of somatic cells (hFSK (46, XY), and hMSC (46, XY)) into a germ cell-like phenotype in vitro. Induced germ cell-like cells showed a marked switch in their transcriptomic profile and expressed several post-meiotic germ line related markers, showed meiotic progression, evidence of epigenetic reprogramming, and approximately 1% were able to complete meiosis as demonstrated by their haploid status and the expression of several post-meiotic markers. Furthermore, xenotransplantation assays demonstrated that a subset of induced cells properly colonize the spermatogonial niche. Knowledge obtained from this work can be used to create in vitro models to study gamete-related diseases in humans. PMID:27112843

  13. Cell adhesion molecules in early chicken embryogenesis.

    PubMed Central

    Thiery, J P; Duband, J L; Rutishauser, U; Edelman, G M

    1982-01-01

    N-CAM, the neural cell adhesion molecule, has been found at a number of regions in the early (1-5 days) chicken embryo by fluorescent antibody techniques. These regions appear to be those concerned with induction of the primary developmental axis (neural plate, neural tube, notochord, somites) or those in which later inductive events occur (neural crest cells, optic, otic, and pharyngeal placodes, cardiac mesoderm, mesonephric primordium, limb buds). The staining patterns in the latter group of regions are highly dynamic and transient and are limited to the epithelial components of the placodes and to the precursors of mesonephric tubules. In neural crest cells, N-CAM appears early, disappears during migration of the cells on fibronectin, and reappears at sites where ganglia are formed. In other regions of the nervous system, particularly those related directly to the neural tube, the N-CAM molecule is stained at all stages. The results raise the possibility that adhesion mediated by N-CAM plays a primary role in early embryogenesis as well as in later histogenesis. Images PMID:6959151

  14. From Somatic Embryo to Synthetic Seed in Citrus spp. Through the Encapsulation Technology.

    PubMed

    Micheli, Maurizio; Standardi, Alvaro

    2016-01-01

    In vitro propagation by somatic embryogenesis represents an efficient alternative method to produce high-quality and healthy plants in Citrus species. The regenerated somatic embryos need protection from mechanical damages during manipulation and transport, as well as nutritive support for their evolution in plantlets after sowing. The encapsulation technology allows to obtain synthetic seeds by covering somatic embryos with a gel of calcium alginate enriched by nutrients. This chapter describes the procedure for producing synthetic seeds containing somatic embryos from different Citrus genotypes. PMID:26619885

  15. Characterization of leafy cotyledon1-like during embryogenesis in Theobroma cacao L.

    PubMed

    Alemanno, Laurence; Devic, Martine; Niemenak, Nicolas; Sanier, Christine; Guilleminot, Jocelyne; Rio, Mariannick; Verdeil, Jean-Luc; Montoro, Pascal

    2008-03-01

    Theobroma cacao L., an economically important crop for developing countries, can be experimentally propagated by somatic embryogenesis. Because of their potential roles in embryogenesis, a gene candidate strategy was initiated to find gene homologues of the members of the leafy cotyledon family of transcription factors. A homologue of the leafy cotyledon1-like gene, that encodes the HAP 3 subunit of the CCAAT box-binding factor, was found in the cocoa genome (TcL1L). The translated peptide shared a high amino acid sequence identity with the homologous genes of Arabidopsis thaliana, Phaseolus coccineus and Helianthus annuus. TcL1L transcripts mainly accumulated in young and immature zygotic embryos, and, to a lesser extent, in young and immature somatic embryos. In situ hybridization specified the localization of the transcripts as being mainly in embryonic cells of young embryos, the meristematic cells of the shoot and root apex of immature embryos, and in the protoderm and epidermis of young and immature embryos, either zygotic or somatic. Non-embryogenic explants did not show TcL1L expression. Ectopic expression of the TcL1L gene could partially rescue the Arabidopsis lec1 mutant phenotype, suggesting a similarity of function in zygotic embryogenesis. PMID:18094994

  16. Somatic embryo mediated mass production of Catharanthus roseus in culture vessel (bioreactor) - A comparative study.

    PubMed

    Mujib, A; Ali, Muzamil; Isah, Tasiu; Dipti

    2014-11-01

    The purpose of this study was to evaluate and compare the use of liquid and solid Murashige and Skoog (MS) medium in different culture vessels for mass production of Catharanthus roseus, an important source of anticancerous compounds, vincristine and vinblastine. Three media conditions i.e. agar-solidified medium (S), liquid medium in agitated conical flask (L) and growtek bioreactor (B) were used. Rapid propagation was achieved through in vitro somatic embryogenesis pathway. The process of embryogenesis has been categorized into induction, proliferation, maturation and germination stages. All in vitro embryogenesis stages were conducted by withdrawing spent liquid medium and by adding fresh MS medium. In optimized 4.52 μM 2,4-D added MS, the callus biomass growth was low in solid (1.65 g) compared to liquid medium in agitated conical flask (1.95 g) and in bioreactor (2.11 g). The number of normal somatic embryos was more in solid medium (99.75/50 mg of callus mass) compared to liquid medium used in conical flask (83.25/callus mass) and growtek bioreactor (84.88/callus mass). The in vitro raised embryos maturated in GA3 (2.60 μM) added medium; and in bioreactor the embryo growth was high, a maximum length of 9.82 mm was observed at the end of four weeks. These embryos germinated into seedlings in BAP (2.22 μM) added medium and the embryo germination ability was more (59.41%) in bioreactor compared to liquid medium in conical flask (55.5%). Shoot length (11.25 mm) was also high in bioreactor compared to agitated conical flask. The liquid medium used in agitated conical flask and bioreactor increased seedling production efficiency, at the same time it also reduced plant recovery time. The embryo generated plants grew normally in outdoor conditions. The exploitation of medium to large culture vessel or bioreactor may make the process more efficient in getting large number of Catharanthus plant as it is the only source of anti-cancerous alkaloids, vincristine and vinblastine. PMID:25313279

  17. Somatic embryo mediated mass production of Catharanthus roseus in culture vessel (bioreactor) – A comparative study

    PubMed Central

    Mujib, A.; Ali, Muzamil; Isah, Tasiu; Dipti

    2014-01-01

    The purpose of this study was to evaluate and compare the use of liquid and solid Murashige and Skoog (MS) medium in different culture vessels for mass production of Catharanthus roseus, an important source of anticancerous compounds, vincristine and vinblastine. Three media conditions i.e. agar-solidified medium (S), liquid medium in agitated conical flask (L) and growtek bioreactor (B) were used. Rapid propagation was achieved through in vitro somatic embryogenesis pathway. The process of embryogenesis has been categorized into induction, proliferation, maturation and germination stages. All in vitro embryogenesis stages were conducted by withdrawing spent liquid medium and by adding fresh MS medium. In optimized 4.52 μM 2,4-D added MS, the callus biomass growth was low in solid (1.65 g) compared to liquid medium in agitated conical flask (1.95 g) and in bioreactor (2.11 g). The number of normal somatic embryos was more in solid medium (99.75/50 mg of callus mass) compared to liquid medium used in conical flask (83.25/callus mass) and growtek bioreactor (84.88/callus mass). The in vitro raised embryos maturated in GA3 (2.60 μM) added medium; and in bioreactor the embryo growth was high, a maximum length of 9.82 mm was observed at the end of four weeks. These embryos germinated into seedlings in BAP (2.22 μM) added medium and the embryo germination ability was more (59.41%) in bioreactor compared to liquid medium in conical flask (55.5%). Shoot length (11.25 mm) was also high in bioreactor compared to agitated conical flask. The liquid medium used in agitated conical flask and bioreactor increased seedling production efficiency, at the same time it also reduced plant recovery time. The embryo generated plants grew normally in outdoor conditions. The exploitation of medium to large culture vessel or bioreactor may make the process more efficient in getting large number of Catharanthus plant as it is the only source of anti-cancerous alkaloids, vincristine and vinblastine. PMID:25313279

  18. vasa and piwi are required for mitotic integrity in early embryogenesis in the spider Parasteatoda tepidariorum.

    PubMed

    Schwager, Evelyn E; Meng, Yue; Extavour, Cassandra G

    2015-06-15

    Studies in vertebrate and invertebrate model organisms on the molecular basis of primordial germ cell (PGC) specification have revealed that metazoans can specify their germ line either early in development by maternally transmitted cytoplasmic factors (inheritance), or later in development by signaling factors from neighboring tissues (induction). Regardless of the mode of PGC specification, once animal germ cells are specified, they invariably express a number of highly conserved genes. These include vasa and piwi, which can play essential roles in any or all of PGC specification, development, or gametogenesis. Although the arthropods are the most speciose animal phylum, to date there have been no functional studies of conserved germ line genes in species of the most basally branching arthropod clade, the chelicerates (which includes spiders, scorpions, and horseshoe crabs). Here we present the first such study by using molecular and functional tools to examine germ line development and the roles of vasa and piwi orthologues in the common house spider Parasteatoda (formerly Achaearanea) tepidariorum. We use transcript and protein expression patterns of Pt-vasa and Pt-piwi to show that primordial germ cells (PGCs) in the spider arise during late embryogenesis. Neither Pt-vasa nor Pt-piwi gene products are localized asymmetrically to any embryonic region before PGCs emerge as paired segmental clusters in opisthosomal segments 2-6 at late germ band stages. RNA interference studies reveal that both genes are required maternally for egg laying, mitotic progression in early embryos, and embryonic survival. Our results add to the growing body of evidence that vasa and piwi can play important roles in somatic development, and provide evidence for a previously hypothesized conserved role for vasa in cell cycle progression. PMID:25257304

  19. SYCHRONIZED SOMATIC EMBRYO DEVELOPMENT IN EMBRYOGENIC SUSPENSIONS OF GRAPEVINE (MUSCADINIA ROTUNDIFOLIA SMALL AND VITIS VINIFERA L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The full advantages of somatic embryogenesis as a regeneration system and essential model for performing functional genomics studies and understanding molecular aspect of the ontogenesis of higher plants are demonstrated only in high-frequency, synchronous embryogenic system in liquid culture. In t...

  20. Linker histone variants control chromatin dynamics during early embryogenesis

    PubMed Central

    Saeki, Hideaki; Ohsumi, Keita; Aihara, Hitoshi; Ito, Takashi; Hirose, Susumu; Ura, Kiyoe; Kaneda, Yasufumi

    2005-01-01

    Complex transitions in chromatin structure produce changes in genome function during development in metazoa. Linker histones, the last component of nucleosomes to be assembled into chromatin, comprise considerably divergent subtypes as compared with core histones. In all metazoa studied, their composition changes dramatically during early embryogenesis concomitant with zygotic gene activation, leading to distinct functional changes that are still poorly understood. Here, we show that early embryonic linker histone B4, which is maternally expressed, is functionally different from somatic histone H1 in influencing chromatin structure and dynamics. We developed a chromatin assembly system with nucleosome assembly protein-1 as a linker histone chaperone. This assay system revealed that maternal histone B4 allows chromatin to be remodeled by ATP-dependent chromatin remodeling factor, whereas somatic histone H1 prevents this remodeling. Structural analysis shows that histone B4 does not significantly restrict the accessibility of linker DNA. These findings define the functional significance of developmental changes in linker histone variants. We propose a model that holds that maternally expressed linker histones are key molecules specifying nuclear dynamics with respect to embryonic totipotency. PMID:15821029

  1. Afferent and efferent specificity in the induction and elicitation of parental cross-protective immunity by an immunogenic murine tumor variant: associative recognition of a unique tumor-specific antigen on somatic cell hybrids.

    PubMed

    LeGrue, S J; Ananthaswamy, H N; Simcik, W J

    1989-09-01

    Highly immunogenic (Imm+) murine tumor cell variants can engender a strong tumor-specific, cross-protective immune response against challenge with the weakly immunogenic parental tumor cell line. We examined the afferent induction and efferent specificity of the parental cross-protective immunity observed following immunization with the Imm+ variant of the murine fibrosarcoma MCA-F, designated MCA-FM1. Specificity of the afferent and efferent responses against the parental tumor in mice immunized with the MCA-FM1 variant were monitored by challenge with the tumor MCA-D, which expresses a tumor-specific antigen that is immunologically distinct from but biochemically related to the MCA-F antigen. We observed that mixture of MCA-D and MCA-FM1 cells at immunization failed to elicit a strong tumor rejection response against challenge with MCA-D. Challenge of MCA-FM1-immune mice with a mixture of MCA-FM1 and MCA-D cells resulted in a significant bystander effect at the site of Imm+ rejection, with reduced growth of the MCA-D tumor. To test the hypothesis that the induction of parental cross-protective immunity required the associative recognition of both the Imm+ neoantigen and the parental tumor antigen on the same cell, we constructed somatic cell hybrids of MCA-D with either MCA-F or MCA-FM1. Surprisingly, the hybrids did not express either parental tumor-specific antigen present on the fusion partners but displayed a unique antigenic specificity designated F/D. Expression of the F/D antigen by both the immunogenic and nonimmunogenic hybrid cell lines demonstrated that the tumor-specific F/D antigen was the focus of the cross-protective immunity. These results demonstrate that associative recognition of the tumor-specific parental antigen with the strongly immunogenic neoantigen coexpressed on the surface of the Imm+ variant is responsible for the afferent induction and efferent elicitation of anti-parental cross-protective immunity. Furthermore, this study is the first to report that the fusion of two syngeneic tumor cell lines reproducibly results in a new tumor antigen specificity at the expense of the original parental specificities. PMID:2474372

  2. A carrot somatic embryo mutant is rescued by chitinase.

    PubMed

    De Jong, A J; Cordewener, J; Lo Schiavo, F; Terzi, M; Vandekerckhove, J; Van Kammen, A; De Vries, S C

    1992-04-01

    At the nonpermissive temperature, somatic embryogenesis of the temperature-sensitive (ts) carrot cell mutant ts11 does not proceed beyond the globular stage. This developmental arrest can be lifted by the addition of proteins secreted by wild-type cells to the culture medium. From this mixture of secreted proteins, a 32-kD glycoprotein, designated extracellular protein 3 (EP3), that allows completion of somatic embryo development in ts11 at the nonpermissive temperature was purified. On the basis of peptide sequences and biochemical characterization, EP3 was identified as a glycosylated acidic endochitinase. The addition of the 32-kD endochitinase to ts11 embryo cultures at the nonpermissive temperature appeared to promote the formation of a correctly formed embryo protoderm. These results imply that a glycosylated acidic endochitinase has an important function in early plant somatic embryo development. PMID:1498601

  3. Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis1[W][OPEN

    PubMed Central

    Huang, Shuanglong; Hill, Robert D.; Wally, Owen S.D.; Dionisio, Giuseppe; Ayele, Belay T.; Jami, Sravan Kumar; Stasolla, Claudio

    2014-01-01

    Programmed cell death (PCD) in multicellular organisms is a vital process in growth, development, and stress responses that contributes to the formation of tissues and organs. Although numerous studies have defined the molecular participants in apoptotic and PCD cascades, successful identification of early master regulators that target specific cells to live or die is limited. Using Zea mays somatic embryogenesis as a model system, we report that the expressions of two plant hemoglobin (Hb) genes (ZmHb1 and ZmHb2) regulate the cell survival/death decision that influences somatic embryogenesis through their cell-specific localization patterns. Suppression of either of the two ZmHbs is sufficient to induce PCD through a pathway initiated by elevated NO and Zn2+ levels and mediated by production of reactive oxygen species. The effect of the death program on the fate of the developing embryos is dependent on the localization patterns of the two ZmHbs. During somatic embryogenesis, ZmHb2 transcripts are restricted to a few cells anchoring the embryos to the subtending embryogenic tissue, whereas ZmHb1 transcripts extend to several embryonic domains. Suppression of ZmHb2 induces PCD in the anchoring cells, allowing the embryos to develop further, whereas suppression of ZmHb1 results in massive PCD, leading to abortion. We conclude that regulation of the expression of these ZmHbs has the capability to determine the developmental fate of the embryogenic tissue during somatic embryogenesis through their effect on PCD. This unique regulation might have implications for development and differentiation in other species. PMID:24784758

  4. The effect of carbohydrates and osmoticum on storage reserve accumulation and germination of Norway spruce somatic embryos.

    PubMed

    Businge, Edward; Bygdell, Joakim; Wingsle, Gunnar; Moritz, Thomas; Egertsdotter, Ulrika

    2013-10-01

    Somatic embryogenesis (SE) represents a useful experimental system for studying the regulatory mechanisms of embryo development. In this study, the effect of carbohydrates and osmoticum on storage reserve accumulation and germination of Norway spruce [Picea abies (L.) Karst] somatic embryos were investigated. Using time lapse photography, we monitored development from proliferation of proembryogenic masses (PEMs) to maturation of somatic embryos in two P. abies cell lines cultured on two maturation treatments. A combination of sugar assays, metabolic and proteomic analyses were used to quantify storage reserves in the mature somatic embryos. The maturation treatment containing a nonpermeating osmoticum, polyethylene glycol (PEG, 7.5%) and maltose (3%) as the carbohydrate gave significantly high maturation and low germination frequencies of somatic embryos compared to the treatment with only 3% sucrose. Somatic embryos treated with 3% sucrose contained high levels of sucrose, raffinose and late embryogenesis abundant (LEA) proteins. These compounds are known to be involved in the acquisition of desiccation tolerance during seed development and maturation. In addition the sucrose treatment significantly increased the content of starch in the somatic embryos while the maltose and PEG treatment resulted in somatic embryos with a high content of storage proteins. The high levels of sucrose, raffinose and LEA proteins in the embryos treated with 3% sucrose suggest that sucrose may improve the germination of somatic embryos by promoting the acquisition of desiccation tolerance. PMID:23421376

  5. Metabolite profiling of somatic embryos of Cyclamen persicum in comparison to zygotic embryos, endosperm, and testa

    PubMed Central

    Winkelmann, Traud; Ratjens, Svenja; Bartsch, Melanie; Rode, Christina; Niehaus, Karsten; Bednarz, Hanna

    2015-01-01

    Somatic embryogenesis has been shown to be an efficient in vitro plant regeneration system for many crops such as the important ornamental plant Cyclamen persicum, for which this regeneration pathway of somatic embryogenesis is of interest for the vegetative propagation of parental lines as well as elite plants. However, somatic embryogenesis is not commercially used in many crops due to several unsolved problems, such as malformations, asynchronous development, deficiencies in maturation and germination of somatic embryos. In contrast, zygotic embryos in seeds develop and germinate without abnormalities in most cases. Instead of time-consuming and labor-intensive experiments involving tests of different in vitro culture conditions and plant growth regulator supplements, we follow a more directed approach. Zygotic embryos served as a reference and were compared to somatic embryos in metabolomic analyses allowing the future optimization of the in vitro system. The aims of this study were to detect differences in the metabolite profiles of torpedo stage somatic and zygotic embryos of C. persicum. Moreover, major metabolites in endosperm and testa were identified and quantified. Two sets of extracts of two to four biological replicates each were analyzed. In total 52 metabolites were identified and quantified in the different tissues. One of the most significant differences between somatic and zygotic embryos was that the proline concentration in the zygotic embryos was about 40 times higher than that found in somatic embryos. Epicatechin, a scavenger for reactive oxygen species, was found in highest abundance in the testa. Sucrose, the most abundant metabolite was detected in significantly higher concentrations in zygotic embryos. Also, a yet unknown trisaccharide, was significantly enriched in zygotic embryos. PMID:26300898

  6. Characterization of cDNAs expressed in the early stages of microspore embryogenesis in barley (Hordeum vulgare) L.

    PubMed

    Vrinten, P L; Nakamura, T; Kasha, K J

    1999-11-01

    To gain insight into the molecular events occurring in the very early stages of barley microspore embryogenesis, cDNA clones corresponding to genes differentially expressed during the early stages of microspore culture were isolated and characterized. A cDNA library established from barley microspores cultured for three days was differentially screened against probes generated from freshly isolated microspores. Three cDNAs representing genes not previously identified in barley were isolated. ECA1 (early culture abundant 1) lacked significant homology to known genes or proteins, and the transcript was only expressed during the early stages of culture. Expression was also reduced in low-density control cultures, therefore this gene may play a role in the early stages of barley microspore embryogenesis. ECGST (early culture glutathione S-transferase) had homology to parA-like genes, which are members of a newly discovered group of glutathione S-transferases (GSTs). The protein corresponding to ECGST may be important in protecting cells from oxidative stress during the culture process. ECLTP (early culture lipid transfer protein) had homology to lipid transfer proteins (LTPs), and had an expression pattern similar to that of an LTP known to be a marker of the early stages of embryogenesis in the carrot somatic embryogenesis system. The identification and characterization of the clones isolated in this study provides new information on the events involved in barley microspore embryogenesis. PMID:10608656

  7. Zygotic embryogenesis in Anthurium (Araceae).

    PubMed

    Matsumoto, T K; Kuehnle, A R; Webb, D T

    1998-11-01

    Morphological, anatomical, and histochemical aspects of zygotic embryogenesis by Anthurium andraeanum Lind. were investigated from 4 to 24 wk postpollination. Anatomical features were correlated with morphology of the spadix and capacity of embryos to germinate in vitro. Development from a single-cell zygote to fully mature seed takes 24 wk. The suspensor was two ranked and obvious during the early stages of embryogeny. It was apparent by week 8, substantial until week 14, and diminished rapidly until its absence by week 22. Differentiation of the shoot apex, cotyledon, and protoderm occurs at 14 wk. The embryo starts to derive nutrition from the endosperm at this time, and germination of cultured ovules reached 56%. By 20 wk the shoot apex had visible leaf primordia and the root apex was clearly defined. The cotyledon was well developed and surrounded the shoot tip. The storage of protein and starch was at its greatest in the endosperm and embryo. Furthermore, 100% germination of cultured ovules and embryos occurred at 20 wk and thereafter. Fully mature embryos at 24 wk are green and contain protoxylem elements. PMID:21680314

  8. Somatization and chronic pain.

    PubMed

    Birket-Smith, M

    2001-10-01

    The experience of pain is related not only to tissue damage and physical illness, but also to mental phenomena including depression, anxiety and somatization. Somatization is common among chronic pain patients and presents special problems in management and treatment. Somatoform patients are often given inappropriate diagnoses, treated for non-existent depressive disorders, and exposed to multiple, superfluous investigations. Psychological models of chronic pain and somatization are presented, and treatment issues including psychotherapy and the use of antidepressants are discussed. PMID:11683662

  9. Somatic symptom disorder

    MedlinePlus

    ... disorders; Somatization disorder; Somatiform disorders; Briquet syndrome; Illness anxiety disorder ... history of abuse. SSD is similar to illness anxiety disorder . This is when a person is overly anxious ...

  10. 14-3-3 isoforms and pattern formation during barley microspore embryogenesis.

    PubMed

    Maraschin, Simone de F; Lamers, Gerda E M; de Pater, B Sylvia; Spaink, Herman P; Wang, Mei

    2003-03-01

    The members of the 14-3-3 isoform family have been shown to be developmentally regulated during animal embryogenesis, where they take part in cell differentiation processes. 14-3-3 isoform-specific expression patterns were studied in plant embryogenic processes, using barley (Hordeum vulgare L.) microspore embryogenesis as a model system. After embryogenesis induction by stress, microspores with enlarged morphology showed higher viability than non-enlarged ones. Following microspore culture, cell division was only observed among the enlarged microspores. Western blot and immunolocalization of three barley 14-3-3 isoforms, 14-3-3A, 14-3-3B and 14-3-3C were carried out using isoform-specific antibodies. The level of 14-3-3C protein was higher in enlarged microspores than in non-enlarged ones. A processed form of 14-3-3A was associated with the death pathway of the non-enlarged microspores. In the early embryogenesis stage, 14-3-3 subcellular localization differed among dividing and non-dividing microspores and the microspore-derived multicellular structures showed a polarized expression pattern of 14-3-3C and a higher 14-3-3A signal in epidermis primordia. In the late embryogenesis stage, 14-3-3C was specifically expressed underneath the L(1) layer of the shoot apical meristem and in the scutellum of embryo-like structures (ELSs). 14-3-3C was also expressed in the scutellum and underneath the L(1) layer of the shoot apical meristem of 21 d after pollination (DAP) zygotic embryos. These results reveal that 14-3-3A processing and 14-3-3C isoform tissue-specific expression are closely related to cell fate and initiation of specific cell type differentiation, providing a new insight into the study of 14-3-3 proteins in plant embryogenesis. PMID:12598573

  11. Mass production of somatic embryos expressing Escherichia coli heat-labile enterotoxin B subunit in Siberian ginseng.

    PubMed

    Kang, Tae-Jin; Lee, Won-Seok; Choi, Eun-Gyung; Kim, Jae-Whune; Kim, Bang-Geul; Yang, Moon-Sik

    2006-01-24

    The B subunit of Escherichia coli heat-labile toxin (LTB) is a potent mucosal immunogen and immunoadjuvant for co-administered antigens. In order to produce large scale of LTB for the development of edible vaccine, we used transgenic somatic embryos of Siberian ginseng, which is known as medicinal plant. When transgenic somatic embryos were cultured in 130L air-lift type bioreactor, they were developed to mature somatic embryos through somatic embryogenesis and contained approximately 0.36% LTB of the total soluble protein. Enzyme-linked immunosorbent assay indicated that the somatic embryo-synthesized LTB protein bound specifically to GM1-ganglioside, suggesting the LTB subunits formed active pentamers. Therefore, the use of the bioreactor system for expression of LTB proteins in somatic embryos allows for continuous mass production in a short-term period. PMID:16174540

  12. Epigenetic and hormonal profile during maturation of Quercus Suber L. somatic embryos.

    PubMed

    Pérez, Marta; Viejo, Marcos; LaCuesta, Maite; Toorop, Peter; Cañal, María Jesús

    2014-09-17

    Somatic embryogenesis is a powerful alternative to conventional mass propagation of Quercus suber L. However, poor quality and incomplete maturation of somatic embryos restrict any application. Given that epigenetic and hormonal control govern many developmental stages, including maturation of zygotic embryos, global DNA methylation and abscisic acid (ABA) were analyzed during development and maturation of cork oak somatic embryos. Our results indicated that development of somatic embryos concurred with a decrease in 5-mdC. In contrast, endogenous ABA content showed a transient increase with a peak in immature E2 embryos denoting the onset of the maturation phase. A cold stratification phase was necessary for embryos to acquire germination ability, which coincided with a significant decrease in 5-mdC and ABA content. Immunohistochemical analyses showed that there was a specific spatial-temporal regulation during embryogenesis, particularly after the cold treatment. The acquisition of germination capacity concurred with a general low 5-mdC signal in the root meristem, while retention of the 5-mdC signal was mainly located in the shoot meristem and provascular tissues. Conversely, ABA immunolocalization was mainly located in the root and shoot apical meristems. Furthermore, a strong decrease in the ABA signal was observed in the root cap after the stratification treatment suggesting a role for the root cap during development of somatic embryos. These results suggest that, in addition to ABA, epigenetic control appears to play an important role for the correct maturation and subsequent germination of cork oak somatic embryos. PMID:25462078

  13. Somatization, Paranoia, and Language.

    ERIC Educational Resources Information Center

    Oxman, Thomas E.; And Others

    1988-01-01

    Free speech of subjects with somatization and paranoia was analyzed to identify and compare self-concept dimensions reflected in their lexical choices. The somatization disorder group conveyed a sense of negativism, distress, and preoccupation with an uncertain self-identity. The paranoid patients portrayed an artificially positive, grandiose…

  14. Microspore embryogenesis in wheat: new marker genes for early, middle and late stages of embryo development.

    PubMed

    Sánchez-Díaz, Rosa Angélica; Castillo, Ana María; Vallés, María Pilar

    2013-09-01

    Microspore embryogenesis involves reprogramming of the pollen immature cell towards embryogenesis. We have identified and characterized a collection of 14 genes induced along different morphological phases of microspore-derived embryo development in wheat (Triticum aestivum L.) anther culture. SERKs and FLAs genes previously associated with somatic embryogenesis and reproductive tissues, respectively, were also included in this analysis. Genes involved in signalling mechanisms such as TaTPD1-like and TAA1b, and two glutathione S-transferase (GSTF2 and GSTA2) were induced when microspores had acquired a 'star-like' morphology or had undergone the first divisions. Genes associated with control of plant development and stress response (TaNF-YA, TaAGL14, TaFLA26, CHI3, XIP-R; Tad1 and WALI6) were activated before exine rupture. When the multicellular structures have been released from the exine, TaEXPB4, TaAGP31-like and an unknown embryo-specific gene TaME1 were induced. Comparison of gene expression, between two wheat cultivars with different response to anther culture, showed that the profile of genes activated before exine rupture was shifted to earlier stages in the low responding cultivar. This collection of genes constitutes a value resource for study mechanism of intra-embryo communication, early pattern formation, cell wall modification and embryo differentiation. PMID:23839308

  15. Piwi regulates Vasa accumulation during embryogenesis in the sea urchin

    PubMed Central

    Yajima, Mamiko; Gustafson, Eric A.; Song, Jia L.; Wessel, Gary M.

    2014-01-01

    Background Piwi proteins are essential for germ line development, stem cell maintenance, and more recently found to function in epigenetic and somatic gene regulation. In the sea urchin Strongylocentrotus purpuratus, two Piwi proteins, Seawi and Piwi-like1, have been identified, yet their functional contributions have not been reported. Result Here we found that Seawi protein was localized uniformly in the early embryo and then became enriched in the primordial germ cells (PGCs) (the small micromere lineage) from blastula stage and thereafter. Morpholino knockdown of Sp-seawi diminished PGC-specific localization of Seawi proteins, and altered expression of other germ line markers such as Vasa and Gustavus, but had no effect on Nanos. Further, Seawi knockdown transiently resulted in Vasa positive cell proliferation in the right coelomic pouch that appear to be derived from the small micromere lineage, yet they quickly disappeared with an indication of apoptosis by larval stage. Severe Seawi knockdown resulted in an increased number of apoptotic cells in the entire gut area. Piwi proteins appear to regulate PGC proliferation perhaps through control of Vasa accumulation. In this organism, Piwi is likely regulating mRNAs, not just transposons, and is potentially functioning both inside and outside of the germ line during embryogenesis. PMID:24218044

  16. Hepatocystin is Essential for TRPM7 Function During Early Embryogenesis

    PubMed Central

    Overton, Jeffrey D.; Komiya, Yuko; Mezzacappa, Courtney; Nama, Kaushik; Cai, Na; Lou, Liping; Fedeles, Sorin V.; Habas, Raymond; Runnels, Loren W.

    2015-01-01

    Mutations in protein kinase C substrate 80K-H (PRKCSH), which encodes for an 80 KDa protein named hepatocystin (80K-H, PRKCSH), gives rise to polycystic liver disease (PCLD). Hepatocystin functions as the noncatalytic beta subunit of Glucosidase II, an endoplasmic reticulum (ER)-resident enzyme involved in processing and quality control of newly synthesized glycoproteins. Patients harboring heterozygous germline mutations in PRKCSH are thought to develop renal cysts as a result of somatic loss of the second allele, which subsequently interferes with expression of the TRP channel polycystin-2 (PKD2). Deletion of both alleles of PRKCSH in mice results in embryonic lethality before embryonic day E11.5. Here, we investigated the function of hepatocystin during Xenopus laevis embryogenesis and identified hepatocystin as a binding partner of the TRPM7 ion channel, whose function is required for vertebrate gastrulation. We find that TRPM7 functions synergistically with hepatocystin. Although other N-glycosylated proteins are critical to early development, overexpression of TRPM7 in Xenopus laevis embryos was sufficient to fully rescue the gastrulation defect caused by loss of hepatocystin. We observed that depletion of hepatocystin in Xenopus laevis embryos decreased TRPM7 expression, indicating that the early embryonic lethality caused by loss of hepatocystin is mainly due to impairment of TRPM7 protein expression. PMID:26671672

  17. Hepatocystin is Essential for TRPM7 Function During Early Embryogenesis.

    PubMed

    Overton, Jeffrey D; Komiya, Yuko; Mezzacappa, Courtney; Nama, Kaushik; Cai, Na; Lou, Liping; Fedeles, Sorin V; Habas, Raymond; Runnels, Loren W

    2015-01-01

    Mutations in protein kinase C substrate 80K-H (PRKCSH), which encodes for an 80 KDa protein named hepatocystin (80K-H, PRKCSH), gives rise to polycystic liver disease (PCLD). Hepatocystin functions as the noncatalytic beta subunit of Glucosidase II, an endoplasmic reticulum (ER)-resident enzyme involved in processing and quality control of newly synthesized glycoproteins. Patients harboring heterozygous germline mutations in PRKCSH are thought to develop renal cysts as a result of somatic loss of the second allele, which subsequently interferes with expression of the TRP channel polycystin-2 (PKD2). Deletion of both alleles of PRKCSH in mice results in embryonic lethality before embryonic day E11.5. Here, we investigated the function of hepatocystin during Xenopus laevis embryogenesis and identified hepatocystin as a binding partner of the TRPM7 ion channel, whose function is required for vertebrate gastrulation. We find that TRPM7 functions synergistically with hepatocystin. Although other N-glycosylated proteins are critical to early development, overexpression of TRPM7 in Xenopus laevis embryos was sufficient to fully rescue the gastrulation defect caused by loss of hepatocystin. We observed that depletion of hepatocystin in Xenopus laevis embryos decreased TRPM7 expression, indicating that the early embryonic lethality caused by loss of hepatocystin is mainly due to impairment of TRPM7 protein expression. PMID:26671672

  18. Ethylene precursors and antagonists increase embryogenesis of Hordeum vulgare L. anther culture.

    PubMed

    Evans, J M; Batty, N P

    1994-09-01

    The role of ethylene in microspore embryogenesis and regeneration was analyzed by studying the effects of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and the ethylene antagonists silver nitrate and silver thiosulphate on the androgenic response of in vitro cultured anthers of seven genotypes of barley. Incorporation of either ACC or silver salts in the culture medium lead to a significant increase in callus induction for five of the seven genotypes tested. The treatment that increased callus induction depended upon genotype. Only anthers cultured on 1 mg l(-1) silver thiosulphate gave rise to fertile plants in all seven genotypes tested. PMID:24193518

  19. Robotic workcell for quality sorting of somatic embryos

    NASA Astrophysics Data System (ADS)

    Chen, F. S.; Ting, K. C.

    1995-01-01

    Somatic embryogenesis is a process considered very promising in mass regeneration of plant materials. Quality evaluation of embryos' viability is deemed necessary during the process. Machine vision techniques have been or are being developed for quality sorting of embryos immediately before germination. In this project, a robotic workcell has been conceptually designed for quality sorting of somatic embryos employing machine vision system. The configuration of the workcell has been modeled using a 3D graphic modeling software package. The workcell component requirements, layout, and materials flow have been investigated for its workability. A numerical model has been developed to simulate the productivity of a workable layout. Some stochastic factors affecting the workcell productivity were considered in the numerical model. Engineering economic analysis was performed on the workcell for evaluating its cost-effectiveness.

  20. In search of markers for somatic embryo maturation in hybrid larch (Larix × eurolepis): global DNA methylation and proteomic analyses.

    PubMed

    Teyssier, Caroline; Maury, Stéphane; Beaufour, Martine; Grondin, Cécile; Delaunay, Alain; Le Metté, Claire; Ader, Kevin; Cadene, Martine; Label, Philippe; Lelu-Walter, Marie-Anne

    2014-02-01

    A global DNA methylation and proteomics approach was used to investigate somatic embryo maturation in hybrid larch. Each developmental step during somatic embryogenesis was associated with a distinct and significantly different global DNA methylation level: from 45.8% mC for undifferentiated somatic embryos (1-week proliferation) to 61.5% mC for immature somatic embryos (1-week maturation), while maturation was associated with a decrease in DNA methylation to 53.4% for mature cotyledonary somatic embryos (8-weeks maturation). The presence of 5-azacytidine (hypo-methylating agent) or hydroxyurea (hyper-methylating agent) in the maturation medium altered the global DNA methylation status of the embryogenic cultures, and significantly reduced both their relative growth rate and embryogenic potential, suggesting an important role for DNA methylation in embryogenesis. Maturation was also assessed by examining changes in the total protein profile. Storage proteins, identified as legumin- and vicilin-like, appeared at the precotyledonary stage. In the proteomic study, total soluble proteins were extracted from embryos after 1 and 8 weeks of maturation, and separated by two-dimensional gel electrophoresis. There were 147 spots which showed significant differences between the stages of maturation; they were found to be involved mainly in primary metabolism and the stabilization of the resulting metabolites. This indicated that the somatic embryo was still metabolically active at 8 weeks of maturation. This is the first report of analyses of global DNA methylation (including the effects of hyper- and hypo-treatments) and proteome during somatic embryogenesis in hybrid larch, and thus provides novel insights into maturation of conifer somatic embryos. PMID:23789891

  1. Somatic symptoms in depression

    PubMed Central

    Kapfhammer, Hans-Peter

    2006-01-01

    Both painful and nonpainful somatic symptoms essentially characterize clinical states of depressive mood. So far, this well-established psychopathological knowledge has been appreciated only insufficiently by the official diagnostic sys-terms of the Diagnostic and Statistical Manual of Mental Disorders, 4th edition, Text Revision (DSM-IVTR) and the ICD-10 Classification of Mental and Behavioral Disorders. Clinical Descriptions and Diagnostic Guidelines (ICD-10). From a perspective of primary care services, this unmet diagnostic need is deplorable, as the main mode of presenting a depression is by reporting somatic symptoms. This somatic form of presentation, however, significantly contributes to low rates of recognition in primary care. A diagnostic challenge may be seen in the differentiation of a depression with prevailing somatic symptoms from anxiety, somatoform disorders, and medical conditions. When somatic symptoms, particularly painful physical conditions, accompany the already debilitating psychiatric and behavioral symptoms of depression, the course of the illness may be more severe, implying a higher risk of early relapse, chronicity suicide, or mortality due to other natural causes, the economic burden increases considerably, the functional status may be hampered heavily, and health-related quality of life may be lowered dramatically. The neurobiological underpinnings of somatic symptoms in depression may guide more promising treatment approaches. PMID:16889108

  2. Isolation and characterization of a diverse set of genes from carrot somatic embryos.

    PubMed Central

    Lin, X; Hwang, G J; Zimmerman, J L

    1996-01-01

    The early events in plant embryogenesis are critical for pattern formation, since it is during this process that the primary apical meristems and the embryo polarity axis are established. However, little is known about the molecular events that are unique to the early stages of embryogenesis. This study of gene expression during plant embryogenesis is focused on identifying molecular markers from carrot (Daucus carota) somatic embryos and characterizing the expression and regulation of these genes through embryo development. A cDNA library, prepared from polysomal mRNA of globular embryos, was screened using a subtracted probe; 49 clones were isolated and preliminarily characterized. Sequence analysis revealed a large set of genes, including many new genes, that are expressed in a variety of patterns during embryogenesis and may be regulated by different molecular mechanisms. To our knowledge, this group of clones represents the largest collection of embryo-enhanced genes isolated thus far, and demonstrates the utility of the subtracted-probe approach to the somatic embryo system. It is anticipated that many of these genes may serve as useful molecular markers for early embryo development. PMID:8938424

  3. Comparative Developmental Staging of Female and Male Water Fleas Daphnia pulex and Daphnia magna During Embryogenesis.

    PubMed

    Toyota, Kenji; Hiruta, Chizue; Ogino, Yukiko; Miyagawa, Shinichi; Okamura, Tetsuro; Onishi, Yuta; Tatarazako, Norihisa; Iguchi, Taisen

    2016-02-01

    The freshwater crustacean genus Daphnia has been used extensively in ecological, developmental and ecotoxicological studies. Daphnids produce only female offspring by parthenogenesis under favorable conditions, but in response to various unfavorable conditions and external stimuli, they produce male offspring. Although we reported that exogenous exposure to juvenile hormones and their analogs can induce male offspring even under female-producing conditions, we recently established a male induction system in the Daphnia pulex WTN6 strain simply by changing day-length. This male and female induction system is suitable for understanding the innate mechanisms of sexual dimorphic development in daphnids. Embryogenesis has been described as a normal plate (developmental staging) in various daphnid species; however, all studies have mainly focused on female development. Here, we describe the developmental staging of both sexes during embryogenesis in two representative daphnids, D. pulex and D. magna, based on microscopic time-course observations. Our findings provide the first detailed insights into male embryogenesis in both species, and contribute to the elucidation of the mechanisms underlying sexual differentiation in daphnids. PMID:26853866

  4. Generation of hermaphrodite transgenic papaya lines with virus resistance via transformation of somatic embryos derived from adventitious roots of in vitro shoots.

    PubMed

    Kung, Yi-Jung; Yu, Tsong-Ann; Huang, Chiung-Huei; Wang, Hui-Chin; Wang, Shin-Lan; Yeh, Shyi-Dong

    2010-08-01

    Papaya production is seriously limited by Papaya ringspot virus (PRSV) worldwide and Papaya leaf-distortion mosaic virus (PLDMV) in Eastern Asia. An efficient transformation method for developing papaya lines with transgenic resistance to these viruses and commercially desirable traits, such as hermaphroditism, is crucial to shorten the breeding program for this fruit crop. In this investigation, an untranslatable chimeric construct pYP08 containing truncated PRSV coat protein (CP) and PLDMV CP genes coupled with the 3' untranslational region of PLDMV, was generated. Root segments from different portions of adventitious roots of in vitro multiple shoots of hermaphroditic plants of papaya cultivars 'Tainung No. 2', 'Sunrise', and 'Thailand' were cultured on induction medium for regeneration into somatic embryos. The highest frequency of somatic embryogenesis was from the root-tip segments of adventitious roots developed 2-4 weeks after rooting in perlite medium. After proliferation, embryogenic tissues derived from somatic embryos were wounded in liquid-phase by carborundum and transformed by Agrobacterium carrying pYP08. Similarly, another construct pBG-PLDMVstop containing untranslatable CP gene of PLDMV was also transferred to 'Sunrise' and 'Thailand', the parental cultivars of 'Tainung No. 2'. Among 107 transgenic lines regenerated from 349 root-tip segments, nine lines of Tainung No. 2 carrying YP08 were highly resistant to PRSV and PLDMV, and 9 lines (8 'Sunrise' and 1 'Thailand') carrying PLDMV CP highly resistant to PLDMV, by a mechanism of post-transcriptional gene silencing. The hermaphroditic characteristics of the transgenic lines were confirmed by PCR with sex-linked primers and phenotypes of flower and fruit. Our approach has generated transgenic resistance to both PRSV and PLDMV with commercially desirable characters and can significantly shorten the time-consuming breeding programs for the generation of elite cultivars of papaya hybrids. PMID:19943109

  5. Periderm prevents pathological epithelial adhesions during embryogenesis.

    PubMed

    Richardson, Rebecca J; Hammond, Nigel L; Coulombe, Pierre A; Saloranta, Carola; Nousiainen, Heidi O; Salonen, Riitta; Berry, Andrew; Hanley, Neil; Headon, Denis; Karikoski, Riitta; Dixon, Michael J

    2014-09-01

    Appropriate development of stratified, squamous, keratinizing epithelia, such as the epidermis and oral epithelia, generates an outer protective permeability barrier that prevents water loss, entry of toxins, and microbial invasion. During embryogenesis, the immature ectoderm initially consists of a single layer of undifferentiated, cuboidal epithelial cells that stratifies to produce an outer layer of flattened periderm cells of unknown function. Here, we determined that periderm cells form in a distinct pattern early in embryogenesis, exhibit highly polarized expression of adhesion complexes, and are shed from the outer surface of the embryo late in development. Mice carrying loss-of-function mutations in the genes encoding IFN regulatory factor 6 (IRF6), I?B kinase-? (IKK?), and stratifin (SFN) exhibit abnormal epidermal development, and we determined that mutant animals exhibit dysfunctional periderm formation, resulting in abnormal intracellular adhesions. Furthermore, tissue from a fetus with cocoon syndrome, a lethal disorder that results from a nonsense mutation in IKKA, revealed an absence of periderm. Together, these data indicate that periderm plays a transient but fundamental role during embryogenesis by acting as a protective barrier that prevents pathological adhesion between immature, adhesion-competent epithelia. Furthermore, this study suggests that failure of periderm formation underlies a series of devastating birth defects, including popliteal pterygium syndrome, cocoon syndrome, and Bartsocas-Papas syndrome. PMID:25133425

  6. Periderm prevents pathological epithelial adhesions during embryogenesis

    PubMed Central

    Richardson, Rebecca J.; Hammond, Nigel L.; Coulombe, Pierre A.; Saloranta, Carola; Nousiainen, Heidi O.; Salonen, Riitta; Berry, Andrew; Hanley, Neil; Headon, Denis; Karikoski, Riitta; Dixon, Michael J.

    2014-01-01

    Appropriate development of stratified, squamous, keratinizing epithelia, such as the epidermis and oral epithelia, generates an outer protective permeability barrier that prevents water loss, entry of toxins, and microbial invasion. During embryogenesis, the immature ectoderm initially consists of a single layer of undifferentiated, cuboidal epithelial cells that stratifies to produce an outer layer of flattened periderm cells of unknown function. Here, we determined that periderm cells form in a distinct pattern early in embryogenesis, exhibit highly polarized expression of adhesion complexes, and are shed from the outer surface of the embryo late in development. Mice carrying loss-of-function mutations in the genes encoding IFN regulatory factor 6 (IRF6), IκB kinase-α (IKKα), and stratifin (SFN) exhibit abnormal epidermal development, and we determined that mutant animals exhibit dysfunctional periderm formation, resulting in abnormal intracellular adhesions. Furthermore, tissue from a fetus with cocoon syndrome, a lethal disorder that results from a nonsense mutation in IKKA, revealed an absence of periderm. Together, these data indicate that periderm plays a transient but fundamental role during embryogenesis by acting as a protective barrier that prevents pathological adhesion between immature, adhesion-competent epithelia. Furthermore, this study suggests that failure of periderm formation underlies a series of devastating birth defects, including popliteal pterygium syndrome, cocoon syndrome, and Bartsocas-Papas syndrome. PMID:25133425

  7. High Efficiency Somatic Embrogenesis and Plant Regeneration in Suspension Cultures of an Ornamental Ginger Hybrid (Hedychium muluense x cv ‘Starburst’)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plants were successfully regenerated via somatic embryogenesis from shoot apex-derived callus of an ornamental ginger hybrid, Hedychium muluense x cv ‘Starburst’. H. muluense is a dwarf species and ‘Starburst’ is a hybrid cultivar with white and very fragrant flowers in a circular, wheel-like arrang...

  8. Two waves of programmed cell death occur during formation and development of somatic embryos in the gymnosperm, Norway spruce.

    PubMed

    Filonova, L H; Bozhkov, P V; Brukhin, V B; Daniel, G; Zhivotovsky, B; von Arnold, S

    2000-12-01

    In the animal life cycle, the earliest manifestations of programmed cell death (PCD) can already be seen during embryogenesis. The aim of this work was to determine if PCD is also involved in the elimination of certain cells during plant embryogenesis. We used a model system of Norway spruce somatic embryogenesis, which represents a multistep developmental pathway with two broad phases. The first phase is represented by proliferating proembryogenic masses (PEMs). The second phase encompasses development of somatic embryos, which arise from PEMs and proceed through the same sequence of stages as described for their zygotic counterparts. Here we demonstrate two successive waves of PCD, which are implicated in the transition from PEMs to somatic embryos and in correct embryonic pattern formation, respectively. The first wave of PCD is responsible for the degradation of PEMs when they give rise to somatic embryos. We show that PCD in PEM cells and embryo formation are closely interlinked processes, both stimulated upon withdrawal or partial depletion of auxins and cytokinins. The second wave of PCD eliminates terminally differentiated embryo-suspensor cells during early embryogeny. During the dismantling phase of PCD, PEM and embryo-suspensor cells exhibit progressive autolysis, resulting in the formation of a large central vacuole. Autolytic degradation of the cytoplasm is accompanied by lobing and budding-like segmentation of the nucleus. Nuclear DNA undergoes fragmentation into both large fragments of about 50 kb and multiples of approximately 180 bp. The tonoplast rupture is delayed until lysis of the cytoplasm and organelles, including the nucleus, is almost complete. The protoplasm then disappears, leaving a cellular corpse represented by only the cell wall. This pathway of cell dismantling suggests overlapping of apoptotic and autophagic types of PCD during somatic embryogenesis in Norway spruce. PMID:11082033

  9. Shusterman on Somatic Experience

    ERIC Educational Resources Information Center

    Maattanen, Pentti

    2010-01-01

    Richard Shusterman's "Body Consciousness" aims at formulating a theory of somaesthetics and somatic experience. There has indeed been a growing interest in the role of the body in experience. Shusterman examines the arguments of six important writers who have been influential in this discussion. The emphasis on the body is natural for a…

  10. Shusterman on Somatic Experience

    ERIC Educational Resources Information Center

    Maattanen, Pentti

    2010-01-01

    Richard Shusterman's "Body Consciousness" aims at formulating a theory of somaesthetics and somatic experience. There has indeed been a growing interest in the role of the body in experience. Shusterman examines the arguments of six important writers who have been influential in this discussion. The emphasis on the body is natural for a

  11. Genetic Regulatory Networks in Embryogenesis and Evolution

    NASA Technical Reports Server (NTRS)

    1998-01-01

    The article introduces a series of papers that were originally presented at a workshop titled Genetic Regulatory Network in Embryogenesis and Evaluation. Contents include the following: evolution of cleavage programs in relationship to axial specification and body plan evolution, changes in cell lineage specification elucidate evolutionary relations in spiralia, axial patterning in the leech: developmental mechanisms and evolutionary implications, hox genes in arthropod development and evolution, heterochronic genes in development and evolution, a common theme for LIM homeobox gene function across phylogeny, and mechanisms of specification in ascidian embryos.

  12. Embryogenesis of brassica rapa l. under clinorotation

    NASA Astrophysics Data System (ADS)

    Popova, A.; Ivanenko, G.

    Investigation of reproductive development of higher plants in spaceflight represents scientific interest first of all with the necessity to work out the plant space technologies for creation of controlled life-support systems. In such systems mainly the higher plants are considered to be an important component that makes it necessary to obtain the several generations of higher plants with their full ontogenesis. As a rule, seeds obtained in three species of the higher plants in a series of experiments differ from the control by some parameters (Merkis, Laurinavichius, 1983; Musgrave et al., 1998; 2000; Levinskikh et all. 1999; Stankovich et al., 2002). It was shown, that immature embryos generated in microgravity were at a range of developmental stage, while the ground control embryos had all reached the premature stage of development (Kuang et al., 2003). Besides, the distinctions in a degree of nutrient substances accumulation in them were revealed (Kuang et al., 2000). Therefore, the elucidation of the possible reasons for distortion of plant reproduction in microgravity demands the further research. In this study we examined embryogenesis of higher plant Brassica rapa L. with an application of slow horizontal clinostats, that allows to deprive the plants the opportunity to perceive the gravitational stimulus. Some plants were clinorotated from the moment sowing of seeds; in other series the experiment plants were placed on clinostats after formation of flower buds. Temporal fixation of the material was used in these experiments, which allow to obtain material for studying of consecutive stages of embryogenesis. The development of 2-21 day-old embryos was studied. Comparative embryological analysis has shown a similarity in the main of process of embryo differentiation produced under clinorotation and in the stationary control. At the early stages of embryogenesis, the distortion in suspensor formation was observed more frequently. Embryos generated in clinorotation variant had a wider range of developmental stages in comparison with the stationary control. At the stage of embryo maturation the various deviations in embryo differentiation were revealed. These distortions were connected both with cotyledon and radicle development. Possible reasons for deviations in the process of embryogenesis in condition of altered gravity are discussed.

  13. Aneuploidy in mammalian somatic cells in vivo.

    PubMed

    Cimino, M C; Tice, R R; Liang, J C

    1986-01-01

    Aneuploidy is an important potential source of human disease and of reproductive failure. Nevertheless, the ability of chemical agents to induce aneuploidy has been investigated only sporadically in intact (whole-animal) mammalian systems. A search of the available literature from the EMCT Aneuploidy File (for years 1970-1983) provided 112 papers that dealt with aneuploidy in mammalian somatic cells in vivo. 59 of these papers did not meet minimal criteria for analysis and were rejected from subsequent review. Of the remaining 53 papers that dealt with aneuploidy induction by chemical agents in mammalian somatic cells in vivo, only 3 (6%) contained data that were considered to be supported conclusively by adequate study designs, execution, and reporting. These 3 papers dealt with 2 chemicals, one of which, mercury, was negative for aneuploidy induction in humans, and the other, pyrimethamine, was positive in an experimental rodent study. The majority of papers (94%) were considered inconclusive for a variety of reasons. The most common reasons for calling a study inconclusive were (a) combining data on hyperploidy with those on hypoploidy and/or polyploidy, (b) an inadequate or unspecified number of animals and/or cells per animal scored per treatment group, and (c) poor data presentation such that animal-to-animal variability could not be assessed. Suggestions for protocol development are made, and the future directions of research into aneuploidy induction are discussed. PMID:3941670

  14. Asymmetric Wolbachia Segregation during Early Brugia malayi Embryogenesis Determines Its Distribution in Adult Host Tissues

    PubMed Central

    Landmann, Frédéric; Foster, Jeremy M.; Slatko, Barton; Sullivan, William

    2010-01-01

    Wolbachia are required for filarial nematode survival and fertility and contribute to the immune responses associated with human filarial diseases. Here we developed whole-mount immunofluorescence techniques to characterize Wolbachia somatic and germline transmission patterns and tissue distribution in Brugia malayi, a nematode responsible for lymphatic filariasis. In the initial embryonic divisions, Wolbachia segregate asymmetrically such that they occupy only a small subset of cells in the developing embryo, facilitating their concentration in the adult hypodermal chords and female germline. Wolbachia are not found in male reproductive tissues and the absence of Wolbachia from embryonic germline precursors in half of the embryos indicates Wolbachia loss from the male germline may occur in early embryogenesis. Wolbachia rely on fusion of hypodermal cells to populate adult chords. Finally, we detect Wolbachia in the secretory canal lumen suggesting living worms may release bacteria and/or their products into their host. PMID:20689574

  15. Somatic embryogenesis of carrot in hormone-free medium: external pH control over morphogenesis

    NASA Technical Reports Server (NTRS)

    Smith, D. L.; Krikorian, A. D.

    1990-01-01

    Cultures of preglobular stage proembryos (PGSPs) were initiated from mechanically wounded mature zygotic embryos of carrot, Daucus carota, on a hormone-free, semisolid medium. These PGSPs have been maintained and multiplied for extended periods without their progression into later embryo stages on the same hormone-free medium containing 1 mM NH4+ as the sole nitrogen source. Sustained maintenance of cultures comprised exclusively of PGSPs was dependent on medium pH throughout the culture period. Best growth and multiplication of PGSP cultures occurred when the pH of unbuffered, hormone-free medium fell from 4.5 to 4 over a 2-week period or when buffered medium was titrated to pH 4. If the hormone-free medium was buffered to sustain a pH at or above 4.5, PGSPs developed into later embryo stages. Maintenance with continuous multiplication of PGSPs occurred equally well on medium containing NH4+ or NH4+ and NO3-, but growth was poor with NO3- alone. Additional observations on the effects of medium components such as various nitrogen sources and levels, sucrose concentration, semisolid supports, type of buffer, borate concentration, activated charcoal, and initial pH that permit optimum maintenance of the PGSPs or foster their continued developmental progression into mature embryos and plantlets are reported. The influence of the pH of the hormone-free medium as a determinant in maintaining cultures as PGSPs or allowing their continued embryonic development are unequivocally demonstrated by gross morphology, scanning electron microscopy, and histological preparations.

  16. Effect of weightlessness conditions on the somatic embryogenesis in the culture of carrot cells

    NASA Technical Reports Server (NTRS)

    Butenko, R. G.; Dmitriyeva, N. N.; Ongko, V.; Basyrova, L. V.

    1977-01-01

    A carrot cell culture seeded in Petri dishes in the United States and transported to the USSR was subjected to weightlessness for 20 days during the flight of Kosmos 782. The controls were cultures placed on a centrifuge (1 g) inside the satellite and cultures left on ground in the U.S.S.R. and the United States. A count of structures in the dishes after the flight showed that the number of developing embryonic structures and the extent of their differentiation in weightlessness did not reliably differ from the number and extent of differentiation in structures developed on the ground. Structures with long roots developed in weightlessness. Analysis of the root zones showed that these roots differed by the increased size of the zone of differentiated cells. The increased size of the zones of differentiated cells can indicate earlier development of embryonic structures.

  17. Specification of the somatic musculature in Drosophila†

    PubMed Central

    Dobi, Krista C.; Schulman, Victoria K.; Baylies, Mary K.

    2015-01-01

    The somatic muscle system formed during Drosophila embryogenesis is required for larvae to hatch, feed, and crawl. This system is replaced in the pupa by a new adult muscle set, responsible for activities such as feeding, walking, and flight. Both the larval and adult muscle systems are comprised of distinct muscle fibers to serve these specific motor functions. In this way, the Drosophila musculature is a valuable model for patterning within a single tissue: while all muscle cells share properties such as the contractile apparatus, properties such as size, position, and number of nuclei are unique for a particular muscle. In the embryo, diversification of muscle fibers relies first on signaling cascades that pattern the mesoderm. Subsequently, the combinatorial expression of specific transcription factors leads muscle fibers to adopt particular sizes, shapes, and orientations. Adult muscle precursors (AMPs), set aside during embryonic development, proliferate during the larval phases and seed the formation of the abdominal, leg, and flight muscles in the adult fly. Adult muscle fibers may either be formed de novo from the fusion of the AMPs, or are created by the binding of AMPs to an existing larval muscle. While less is known about adult muscle specification compared to the larva, expression of specific transcription factors is also important for its diversification. Increasingly, the mechanisms required for the diversification of fly muscle have found parallels in vertebrate systems and mark Drosophila as a robust model system to examine questions about how diverse cell types are generated within an organism. PMID:25728002

  18. Selection of Norway spruce somatic embryos by computer vision

    NASA Astrophysics Data System (ADS)

    Hamalainen, Jari J.; Jokinen, Kari J.

    1993-05-01

    A computer vision system was developed for the classification of plant somatic embryos. The embryos are in a Petri dish that is transferred with constant speed and they are recognized as they pass a line scan camera. A classification algorithm needs to be installed for every plant species. This paper describes an algorithm for the recognition of Norway spruce (Picea abies) embryos. A short review of conifer micropropagation by somatic embryogenesis is also given. The recognition algorithm is based on features calculated from the boundary of the object. Only part of the boundary corresponding to the developing cotyledons (2 - 15) and the straight sides of the embryo are used for recognition. An index of the length of the cotyledons describes the developmental stage of the embryo. The testing set for classifier performance consisted of 118 embryos and 478 nonembryos. With the classification tolerances chosen 69% of the objects classified as embryos by a human classifier were selected and 31$% rejected. Less than 1% of the nonembryos were classified as embryos. The basic features developed can probably be easily adapted for the recognition of other conifer somatic embryos.

  19. Somatization disorders in dermatology.

    PubMed

    Gupta, Madhulika A

    2006-02-01

    This paper reviews a wide range of somatization-related symptoms that are encountered in dermatology. These include the unexplained cutaneous sensory syndromes especially the cutaneous dysesthesias associated with pain, numbness and pruritus; traumatic memories in post-traumatic stress disorder (PTSD) which are experienced on a sensory level as 'body memories' and may present as local or generalized pruritic states, urticaria and angioedema; and unexplained flushing reactions and profuse perspiration, in addition to unexplained exacerbations of stress-reactive dermatoses such as psoriasis and atopic eczema secondary to the autonomic hyperarousal in PTSD; classic 'pseudoneurologic' symptoms associated with dissociation including unexplained loss of touch or pain, in addition to the self-induced dermatoses such as dermatitis artefacta and trichotillomania that are encountered with dissociative states; and body dysmorphic disorder where the patient often presents with a somatic preoccupation involving the skin or hair. PMID:16451879

  20. Reprogramming of somatic cells.

    PubMed

    Rajasingh, Johnson

    2012-01-01

    Reprogramming of adult somatic cells into pluripotent stem cells may provide an attractive source of stem cells for regenerative medicine. It has emerged as an invaluable method for generating patient-specific stem cells of any cell lineage without the use of embryonic stem cells. A revolutionary study in 2006 showed that it is possible to convert adult somatic cells directly into pluripotent stem cells by using a limited number of pluripotent transcription factors and is called as iPS cells. Currently, both genomic integrating viral and nonintegrating nonviral methods are used to generate iPS cells. However, the viral-based technology poses increased risk of safety, and more studies are now focused on nonviral-based technology to obtain autologous stem cells for clinical therapy. In this review, the pros and cons of the present iPS cell technology and the future direction for the successful translation of this technology into the clinic are discussed. PMID:22917226

  1. AID AND SOMATIC HYPERMUTATION

    PubMed Central

    Maul, Robert W.; Gearhart, Patricia J.

    2010-01-01

    In response to an assault by foreign organisms, peripheral B cells can change their antibody affinity and isotype by somatically mutating their genomic DNA. The ability of a cell to modify its DNA is exceptional in light of the potential consequences of genetic alterations to cause human disease and cancer. Thus, as expected, this mechanism of antibody diversity is tightly regulated and coordinated through one protein, activation induced deaminase (AID). AID produces diversity by converting cytosine to uracil within the immunoglobulin loci. The deoxyuracil residue is mutagenic when paired with deoxyguanosine, since it mimics thymidine during DNA replication. Additionally, B cells can manipulate the DNA repair pathways so that deoxyuracils are not faithfully repaired. Therefore, an intricate balance exists which is regulated at multiple stages to promote mutation of immunoglobulin genes, while retaining integrity of the rest of the genome. Here we discuss and summarize the current understanding of how AID functions to cause somatic hypermutation. PMID:20510733

  2. Hormonal responses during early embryogenesis in maize.

    PubMed

    Chen, Junyi; Lausser, Andreas; Dresselhaus, Thomas

    2014-04-01

    Plant hormones have been shown to regulate key processes during embryogenesis in the model plant Arabidopsis thaliana, but the mechanisms that determine the peculiar embryo pattern formation of monocots are largely unknown. Using the auxin and cytokinin response markers DR5 and TCSv2 (two-component system, cytokinin-responsive promoter version #2), as well as the auxin efflux carrier protein PIN1a (PINFORMED1a), we have studied the hormonal response during early embryogenesis (zygote towards transition stage) in the model and crop plant maize. Compared with the hormonal response in Arabidopsis, we found that detectable hormone activities inside the developing maize embryo appeared much later. Our observations indicate further an important role of auxin, PIN1a and cytokinin in endosperm formation shortly after fertilization. Apparent auxin signals within adaxial endosperm cells and cytokinin responses in the basal endosperm transfer layer as well as chalazal endosperm are characteristic for early seed development in maize. Moreover, auxin signalling in endosperm cells is likely to be involved in exogenous embryo patterning as auxin responses in the endosperm located around the embryo proper correlate with adaxial embryo differentiation and outgrowth. Overall, the comparison between Arabidopsis and maize hormone response and flux suggests intriguing mechanisms in monocots that are used to direct their embryo patterning, which is significantly different from that of eudicots. PMID:24646239

  3. Late Embryogenesis Abundant (LEA) proteins in legumes.

    PubMed

    Battaglia, Marina; Covarrubias, Alejandra A

    2013-01-01

    Plants are exposed to different external conditions that affect growth, development, and productivity. Water deficit is one of these adverse conditions caused by drought, salinity, and extreme temperatures. Plants have developed different responses to prevent, ameliorate or repair the damage inflicted by these stressful environments. One of these responses is the activation of a set of genes encoding a group of hydrophilic proteins that typically accumulate to high levels during seed dehydration, at the last stage of embryogenesis, hence named Late Embryogenesis Abundant (LEA) proteins. LEA proteins also accumulate in response to water limitation in vegetative tissues, and have been classified in seven groups based on their amino acid sequence similarity and on the presence of distinctive conserved motifs. These proteins are widely distributed in the plant kingdom, from ferns to angiosperms, suggesting a relevant role in the plant response to this unfavorable environmental condition. In this review, we analyzed the LEA proteins from those legumes whose complete genomes have been sequenced such as Phaseolus vulgaris, Glycine max, Medicago truncatula, Lotus japonicus, Cajanus cajan, and Cicer arietinum. Considering their distinctive motifs, LEA proteins from the different groups were identified, and their sequence analysis allowed the recognition of novel legume specific motifs. Moreover, we compile their transcript accumulation patterns based on publicly available data. In spite of the limited information on these proteins in legumes, the analysis and data compiled here confirm the high correlation between their accumulation and water deficit, reinforcing their functional relevance under this detrimental conditions. PMID:23805145

  4. Anisotropic growth shapes intestinal tissues during embryogenesis

    PubMed Central

    Ben Amar, Martine; Jia, Fei

    2013-01-01

    Embryogenesis offers a real laboratory for pattern formation, buckling, and postbuckling induced by growth of soft tissues. Each part of our body is structured in multiple adjacent layers: the skin, the brain, and the interior of organs. Each layer has a complex biological composition presenting different elasticity. Generated during fetal life, these layers will experience growth and remodeling in the early postfertilization stages. Here, we focus on a herringbone pattern occurring in fetal intestinal tissues. Common to many mammalians, this instability is a precursor of the villi, finger-like projections into the lumen. For avians (chicks’ and turkeys’ embryos), it has been shown that, a few days after fertilization, the mucosal epithelium of the duodenum is smooth, and then folds emerge, which present 2 d later a pronounced zigzag instability. Many debates and biological studies are devoted to this specific morphology, which regulates the cell renewal in the intestine. After reviewing experimental results about duodenum morphogenesis, we show that a model based on simplified hypothesis for the growth of the mesenchyme can explain buckling and postbuckling instabilities. Being completely analytical, it is based on biaxial compressive stresses due to differential growth between layers and it predicts quantitatively the morphological changes. The growth anisotropy increasing with time, the competition between folds and zigzags, is proved to occur as a secondary instability. The model is compared with available experimental data on chick’s duodenum and can be applied to other intestinal tissues, the zigzag being a common and spectacular microstructural pattern of intestine embryogenesis. PMID:23754398

  5. Embryogenesis: Pattern Formation from a Single Cell

    PubMed Central

    Capron, Arnaud; Chatfield, Steven; Provart, Nicholas; Berleth, Thomas

    2009-01-01

    During embryogenesis a single cell gives rise to a functional multicellular organism. In higher plants, as in many other multicellular systems, essential architectural features, such as body axes and major tissue layers are established early in embryogenesis and serve as a positional framework for subsequent pattern elaboration. In Arabidopsis, the apicalbasal axis and the radial pattern of tissues wrapped around it are already recognizable in young embryos of only about a hundred cells in size. This early axial pattern seems to provide a coordinate system for the embryonic initiation of shoot and root. Findings from genetic studies in Arabidopsis are revealing molecular mechanisms underlying the initial establishment of the axial core pattern and its subsequent elaboration into functional shoots and roots. The genetic programs operating in the early embryo organize functional cell patterns rapidly and reproducibly from minimal cell numbers. Understanding their molecular details could therefore greatly expand our ability to generate plant body patterns de novo, with important implications for plant breeding and biotechnology. PMID:22303250

  6. Somatic sex determination.

    PubMed Central

    Zarkower, David

    2006-01-01

    C. elegans occurs in two natural sexes, the XX hermaphrodite and the XO male, which differ extensively in anatomy, physiology, and behavior. All somatic differences between the sexes result from the differential activity of a "global" sex determination regulatory pathway. This pathway also controls X chromosome dosage compensation, which is coordinated with sex determination by the action of the three SDC proteins. The SDC proteins control somatic and germline sex by transcriptional repression of the her-1 gene. HER-1 is a secreted protein that controls a regulatory module consisting of a transmembrane receptor, TRA-2, three intracellular FEM proteins, and the zinc finger transcription factor TRA-1. The molecular workings of this regulatory module are still being elucidated. Similarity of TRA-2 to patched receptors and of TRA-1 to GLI proteins suggests that parts of the global pathway originally derived from a Hedgehog signaling pathway. TRA-1 controls all aspects of somatic sexual differentiation, presumably by regulating a variety of tissue- and cell-specific downstream targets, including the cell death regulator EGL-1 and the male sexual regulator MAB-3. Sex determination evolves rapidly, and conservation of sexual regulators between phyla has been elusive. An apparent exception involves DM domain proteins, including MAB-3, which control sexual differentiation in nematodes, arthropods, and vertebrates. Important issues needing more study include the detailed molecular mechanisms of the global pathway, the identities of additional sexual regulators acting in the global pathway and downstream of TRA-1, and the evolutionary history of the sex determination pathway. Recently developed genetic and genomic technologies and comparative studies in divergent species have begun to address these issues. PMID:18050479

  7. 5-azacytidine promotes microspore embryogenesis initiation by decreasing global DNA methylation, but prevents subsequent embryo development in rapeseed and barley

    PubMed Central

    Solís, María-Teresa; El-Tantawy, Ahmed-Abdalla; Cano, Vanesa; Risueño, María C.; Testillano, Pilar S.

    2015-01-01

    Microspores are reprogrammed by stress in vitro toward embryogenesis. This process is an important tool in breeding to obtain double-haploid plants. DNA methylation is a major epigenetic modification that changes in differentiation and proliferation. We have shown changes in global DNA methylation during microspore reprogramming. 5-Azacytidine (AzaC) cannot be methylated and leads to DNA hypomethylation. AzaC is a useful demethylating agent to study DNA dynamics, with a potential application in microspore embryogenesis. This work analyzes the effects of short and long AzaC treatments on microspore embryogenesis initiation and progression in two species, the dicot Brassica napus and the monocot Hordeum vulgare. This involved the quantitative analyses of proembryo and embryo production, the quantification of DNA methylation, 5-methyl-deoxy-cytidine (5mdC) immunofluorescence and confocal microscopy, and the analysis of chromatin organization (condensation/decondensation) by light and electron microscopy. Four days of AzaC treatments (2.5 μM) increased embryo induction, response associated with a decrease of DNA methylation, modified 5mdC, and heterochromatin patterns compared to untreated embryos. By contrast, longer AzaC treatments diminished embryo production. Similar effects were found in both species, indicating that DNA demethylation promotes microspore reprogramming, totipotency acquisition, and embryogenesis initiation, while embryo differentiation requires de novo DNA methylation and is prevented by AzaC. This suggests a role for DNA methylation in the repression of microspore reprogramming and possibly totipotency acquisition. Results provide new insights into the role of epigenetic modifications in microspore embryogenesis and suggest a potential benefit of inhibitors, such as AzaC, to improve the process efficiency in biotechnology and breeding programs. PMID:26161085

  8. 5-azacytidine promotes microspore embryogenesis initiation by decreasing global DNA methylation, but prevents subsequent embryo development in rapeseed and barley.

    PubMed

    Solís, María-Teresa; El-Tantawy, Ahmed-Abdalla; Cano, Vanesa; Risueño, María C; Testillano, Pilar S

    2015-01-01

    Microspores are reprogrammed by stress in vitro toward embryogenesis. This process is an important tool in breeding to obtain double-haploid plants. DNA methylation is a major epigenetic modification that changes in differentiation and proliferation. We have shown changes in global DNA methylation during microspore reprogramming. 5-Azacytidine (AzaC) cannot be methylated and leads to DNA hypomethylation. AzaC is a useful demethylating agent to study DNA dynamics, with a potential application in microspore embryogenesis. This work analyzes the effects of short and long AzaC treatments on microspore embryogenesis initiation and progression in two species, the dicot Brassica napus and the monocot Hordeum vulgare. This involved the quantitative analyses of proembryo and embryo production, the quantification of DNA methylation, 5-methyl-deoxy-cytidine (5mdC) immunofluorescence and confocal microscopy, and the analysis of chromatin organization (condensation/decondensation) by light and electron microscopy. Four days of AzaC treatments (2.5 μM) increased embryo induction, response associated with a decrease of DNA methylation, modified 5mdC, and heterochromatin patterns compared to untreated embryos. By contrast, longer AzaC treatments diminished embryo production. Similar effects were found in both species, indicating that DNA demethylation promotes microspore reprogramming, totipotency acquisition, and embryogenesis initiation, while embryo differentiation requires de novo DNA methylation and is prevented by AzaC. This suggests a role for DNA methylation in the repression of microspore reprogramming and possibly totipotency acquisition. Results provide new insights into the role of epigenetic modifications in microspore embryogenesis and suggest a potential benefit of inhibitors, such as AzaC, to improve the process efficiency in biotechnology and breeding programs. PMID:26161085

  9. Comparative proteomic analysis of somatic embryo maturation in Carica papaya L.

    PubMed Central

    2014-01-01

    Background Somatic embryogenesis is a complex process regulated by numerous factors. The identification of proteins that are differentially expressed during plant development could result in the development of molecular markers of plant metabolism and provide information contributing to the monitoring and understanding of different biological responses. In addition, the identification of molecular markers could lead to the optimization of protocols allowing the use of biotechnology for papaya propagation and reproduction. This work aimed to investigate the effects of polyethylene glycol (PEG) on somatic embryo development and the protein expression profile during somatic embryo maturation in papaya (Carica papaya L.). Results The maturation treatment supplemented with 6% PEG (PEG6) resulted in the greatest number of somatic embryos and induced differential protein expression compared with cultures grown under the control treatment. Among 135 spots selected for MS/MS analysis, 76 spots were successfully identified, 38 of which were common to both treatments, while 14 spots were unique to the control treatment, and 24 spots were unique to the PEG6 treatment. The identified proteins were assigned to seven categories or were unclassified. The most representative class of proteins observed in the control treatment was associated with the stress response (25.8%), while those under PEG6 treatment were carbohydrate and energy metabolism (18.4%) and the stress response (18.4%). Conclusions The differential expression of three proteins (enolase, esterase and ADH3) induced by PEG6 treatment could play an important role in maturation, and these proteins could be characterized as candidate biomarkers of somatic embryogenesis in papaya. PMID:25076862

  10. The use of acetyl-CoA carboxylase activity and changes in wall composition as measures of embryogenesis in tissue cultures of oil palm (Elaeis guineensis).

    PubMed Central

    Turnham, E; Northcote, D H

    1982-01-01

    With some lines of oil-palm tissue cultures embryogenesis occurs spontaneously within the callus grown on a medium containing 2.5 mg of 3-naphthylacetic acid/litre. One of the initial biochemical events that occurs just before the embryoid can be seen is the accumulation of fat droplets within the cells. This accumulation of lipid is correlated with an increase in acetyl-CoA carboxylase activity. The carboxylase is thus probably a rate-limiting step in fatty acid synthesis in these cells and can be used as a quantitative marker of somatic embryogenesis within the tissue. During the development of the embryoid tissue there is an increase in cell division and the differentiation of vascular cells with secondary thickened walls. These stages of the differentiation may be monitored by measuring the ratio of pectin synthesis (polygalacturonic acid formation) to hemicellulose synthesis (xylan formation). Images PLATE 1 PMID:6130759

  11. Somatic mosaicism and variable expressivity.

    PubMed

    Gottlieb, B; Beitel, L K; Trifiro, M A

    2001-02-01

    For more than 50 years geneticists have assumed that variations in phenotypic expression are caused by alterations in genotype. Recent evidence shows that 'simple' mendelian disorders or monogenic traits are often far from simple, exhibiting phenotypic variation (variable expressivity) that cannot be explained entirely by a gene or allelic alteration. In certain cases of androgen insensitivity syndrome caused by identical mutations in the androgen receptor gene, phenotypic variability is caused by somatic mosaicism, that is, somatic mutations that occur only in certain androgen-sensitive cells. Recently, more than 30 other genetic conditions that exhibit variable expressivity have been linked to somatic mosaicism. Somatic mutations have also been identified in diseases such as prostate and colorectal cancer. Therefore, the concept of somatic mutations and mosaicism is likely to have far reaching consequences for genetics, in particular in areas such as genetic counseling. PMID:11173116

  12. High Genetic and Epigenetic Stability in Coffea arabica Plants Derived from Embryogenic Suspensions and Secondary Embryogenesis as Revealed by AFLP, MSAP and the Phenotypic Variation Rate

    PubMed Central

    Bobadilla Landey, Roberto; Cenci, Alberto; Georget, Frédéric; Bertrand, Benoît; Camayo, Gloria; Dechamp, Eveline; Herrera, Juan Carlos; Santoni, Sylvain; Lashermes, Philippe; Simpson, June; Etienne, Hervé

    2013-01-01

    Embryogenic suspensions that involve extensive cell division are risky in respect to genome and epigenome instability. Elevated frequencies of somaclonal variation in embryogenic suspension-derived plants were reported in many species, including coffee. This problem could be overcome by using culture conditions that allow moderate cell proliferation. In view of true-to-type large-scale propagation of C. arabica hybrids, suspension protocols based on low 2,4-D concentrations and short proliferation periods were developed. As mechanisms leading to somaclonal variation are often complex, the phenotypic, genetic and epigenetic changes were jointly assessed so as to accurately evaluate the conformity of suspension-derived plants. The effects of embryogenic suspensions and secondary embryogenesis, used as proliferation systems, on the genetic conformity of somatic embryogenesis-derived plants (emblings) were assessed in two hybrids. When applied over a 6 month period, both systems ensured very low somaclonal variation rates, as observed through massive phenotypic observations in field plots (0.74% from 200 000 plant). Molecular AFLP and MSAP analyses performed on 145 three year-old emblings showed that polymorphism between mother plants and emblings was extremely low, i.e. ranges of 0–0.003% and 0.07–0.18% respectively, with no significant difference between the proliferation systems for the two hybrids. No embling was found to cumulate more than three methylation polymorphisms. No relation was established between the variant phenotype (27 variants studied) and a particular MSAP pattern. Chromosome counting showed that 7 of the 11 variant emblings analyzed were characterized by the loss of 1–3 chromosomes. This work showed that both embryogenic suspensions and secondary embryogenesis are reliable for true-to-type propagation of elite material. Molecular analyses revealed that genetic and epigenetic alterations are particularly limited during coffee somatic embryogenesis. The main change in most of the rare phenotypic variants was aneuploidy, indicating that mitotic aberrations play a major role in somaclonal variation in coffee. PMID:23418563

  13. Expression of developmental genes during early embryogenesis of Hydra.

    PubMed

    Fröbius, Andreas C; Genikhovich, Gregory; Kürn, Ulrich; Anton-Erxleben, Friederike; Bosch, Thomas C G

    2003-09-01

    Hydra is a classical model to study key features of embryogenesis such as axial patterning and stem cell differentiation. In contrast to other organisms where these mechanisms are active only during embryonic development, in Hydra they can be studied in adults. The underlying assumption is that the machinery governing adult patterning mimics regulatory mechanisms which are also active during early embryogenesis. Whether, however, Hydra embryogenesis is governed by the same mechanisms which are controlling adult patterning, remains to be shown. In this paper, in precisely staged Hydra embryos, we examined the expression pattern of 15 regulatory genes shown previously to play a role in adult patterning and cell differentiation. RT-PCR revealed that most of the genes examined were expressed in rather late embryonic stages. In situ hybridization, nuclear run-on experiments, and staining of nucleolar organizer region-associated proteins indicated that genes expressed in early embryos are transcribed in the engulfed "nurse cells" (endocytes). This is the first direct evidence that endocytes in Hydra not only provide nutrients to the developing oocyte but also produce maternal factors critical for embryogenesis. Our findings are an initial step towards understanding the molecular machinery controlling embryogenesis of a key group of basal metazoans and raise the possibility that in Hydra there are differences in the mechanisms controlling embryogenesis and adult patterning. PMID:12883882

  14. Environmental magnetic fields: Influences on early embryogenesis

    SciTech Connect

    Cameron, I.L.; Hardman, W.E.; Winters, W.D.; Zimmerman, S.; Zimmerman, A.M. )

    1993-04-01

    A 10-mG, 50 to 60-Hz magnetic field is in the intensity and frequency range that people worldwide are often exposed to in homes and in the workplace. Studies about the effects of 50- to 100-Hz electromagnetic fields on various species of animal embryos (fish, chick, fly, sea urchin, rat, and mouse) indicate that early stages of embryonic development are responsive to fluctuating magnetic fields. Chick, sea urchin, and mouse embryos are responsive to magnetic field intensities of 10-100 mG. Results from studies on sea urchin embryos indicate that exposure to conditions of rotating 60-Hz magnetic fields, e.g., similar to those in our environment, interferes with cell proliferation at the morula stage in a manner dependent on field intensity. The cleavage stages, prior to the 64-cell stage, were not delayed by this rotating 60-Hz magnetic field suggesting that the ionic surges, DNA replication, and translational events essential for early cleavage stages were not significantly altered. Studies of histone synthesis in early sea urchin embryos indicated that the rotating 60-Hz magnetic field decreased zygotic expression of early histone genes at the morula stage and suggests that this decrease in early histone production was limiting to cell proliferation. Whether these comparative observations from animal development studies will be paralleled by results from studies of human embryogenesis, as suggested by some epidemiology studies, has yet to be established. 38 refs.

  15. Gene expression throughout a vertebrate's embryogenesis

    PubMed Central

    2011-01-01

    Background Describing the patterns of gene expression during embryonic development has broadened our understanding of the processes and patterns that define morphogenesis. Yet gene expression patterns have not been described throughout vertebrate embryogenesis. This study presents statistical analyses of gene expression during all 40 developmental stages in the teleost Fundulus heteroclitus using four biological replicates per stage. Results Patterns of gene expression for 7,000 genes appear to be important as they recapitulate developmental timing. Among the 45% of genes with significant expression differences between pairs of temporally adjacent stages, significant differences in gene expression vary from as few as five to more than 660. Five adjacent stages have disproportionately more significant changes in gene expression (> 200 genes) relative to other stages: four to eight and eight to sixteen cell stages, onset of circulation, pre and post-hatch, and during complete yolk absorption. The fewest differences among adjacent stages occur during gastrulation. Yet, at stage 16, (pre-mid-gastrulation) the largest number of genes has peak expression. This stage has an over representation of genes in oxidative respiration and protein expression (ribosomes, translational genes and proteases). Unexpectedly, among all ribosomal genes, both strong positive and negative correlations occur. Similar correlated patterns of expression occur among all significant genes. Conclusions These data provide statistical support for the temporal dynamics of developmental gene expression during all stages of vertebrate development. PMID:21356103

  16. Classification of plant somatic embryos by computer vision.

    PubMed

    Hämäläinen, J J; Kurtén, U; Kauppinen, V

    1993-01-01

    This article deals with the automation of the process of somatic embryogenesis for the propagation of plants. An important problem is the monitoring of the embryo production process in order to decide the time to start harvesting embryos for further processing. The classification algorithm development for somatic embryos of birch (Betula pendula Roth) showed that automated recognition of embryos at different developmental stages is possible. No globular stage embryos were classified to be heart or torpedo stage and no heart or torpedo stage embryos were classified to be at globular stage. Heart and torpedo stage embryos were classified into three developmental classes by a new index that describes the relation of embryo breadth to the length of the root. The probability of classifying a nonembryo as an embryo was less than 1%, and 14% of the object classified as embryos by a human expert were discarded by the algorithm. A computer vision system suitable for automated monitoring of samples from the bioreactor was constructed. PMID:18601243

  17. DNA sequences that activate isocitrate lyase gene expression during late embryogenesis and during postgerminative growth.

    PubMed Central

    Zhang, J Z; Santes, C M; Engel, M L; Gasser, C S; Harada, J J

    1996-01-01

    We analyzed DNA sequences that regulate the expression of an isocitrate lyase gene from Brassica napus L. during late embryogenesis and during postgerminative growth to determine whether glyoxysomal function is induced by a common mechanism at different developmental stages. beta-Glucuronidase constructs were used both in transient expression assays in B. napus and in transgenic Arabidopsis thaliana to identify the segments of the isocitrate lyase 5' flanking region that influence promoter activity. DNA sequences that play the principal role in activating the promoter during post-germinative growth are located more than 1,200 bp upstream of the gene. Distinct DNA sequences that were sufficient for high-level expression during late embryogenesis but only low-level expression during postgerminative growth were also identified. Other parts of the 5' flanking region increased promoter activity both in developing seed and in seedlings. We conclude that a combination of elements is involved in regulating the isocitrate lyase gene and that distinct DNA sequences play primary roles in activating the gene in embryos and in seedlings. These findings suggest that different signals contribute to the induction of glyoxysomal function during these two developmental stages. We also showed that some of the constructs were expressed differently in transient expression assays and in transgenic plants. PMID:8934622

  18. Epigenetic predisposition to reprogramming fates in somatic cells

    PubMed Central

    Pour, Maayan; Pilzer, Inbar; Rosner, Roni; Smith, Zachary D; Meissner, Alexander; Nachman, Iftach

    2015-01-01

    Reprogramming to pluripotency is a low-efficiency process at the population level. Despite notable advances to molecularly characterize key steps, several fundamental aspects remain poorly understood, including when the potential to reprogram is first established. Here, we apply live-cell imaging combined with a novel statistical approach to infer when somatic cells become fated to generate downstream pluripotent progeny. By tracing cell lineages from several divisions before factor induction through to pluripotent colony formation, we find that pre-induction sister cells acquire similar outcomes. Namely, if one daughter cell contributes to a lineage that generates induced pluripotent stem cells (iPSCs), its paired sibling will as well. This result suggests that the potential to reprogram is predetermined within a select subpopulation of cells and heritable, at least over the short term. We also find that expanding cells over several divisions prior to factor induction does not increase the per-lineage likelihood of successful reprogramming, nor is reprogramming fate correlated to neighboring cell identity or cell-specific reprogramming factor levels. By perturbing the epigenetic state of somatic populations with Ezh2 inhibitors prior to factor induction, we successfully modulate the fraction of iPSC-forming lineages. Our results therefore suggest that reprogramming potential may in part reflect preexisting epigenetic heterogeneity that can be tuned to alter the cellular response to factor induction. PMID:25600117

  19. Genes encoding novel secreted and transmembrane proteins are temporally and spatially regulated during Drosophila melanogaster embryogenesis

    PubMed Central

    Zúñiga, Alejandro; Hödar, Christian; Hanna, Patricia; Ibáñez, Freddy; Moreno, Pablo; Pulgar, Rodrigo; Pastenes, Luis; González, Mauricio; Cambiazo, Verónica

    2009-01-01

    Background Morphogenetic events that shape the Drosophila melanogaster embryo are tightly controlled by a genetic program in which specific sets of genes are up-regulated. We used a suppressive subtractive hybridization procedure to identify a group of developmentally regulated genes during early stages of D. melanogaster embryogenesis. We studied the spatiotemporal activity of these genes in five different intervals covering 12 stages of embryogenesis. Results Microarrays were constructed to confirm induction of expression and to determine the temporal profile of isolated subtracted cDNAs during embryo development. We identified a set of 118 genes whose expression levels increased significantly in at least one developmental interval compared with a reference interval. Of these genes, 53% had a phenotype and/or molecular function reported in the literature, whereas 47% were essentially uncharacterized. Clustering analysis revealed demarcated transcript groups with maximum gene activity at distinct developmental intervals. In situ hybridization assays were carried out on 23 uncharacterized genes, 15 of which proved to have spatiotemporally restricted expression patterns. Among these 15 uncharacterized genes, 13 were found to encode putative secreted and transmembrane proteins. For three of them we validated our protein sequence predictions by expressing their cDNAs in Drosophila S2R+ cells and analyzed the subcellular distribution of recombinant proteins. We then focused on the functional characterization of the gene CG6234. Inhibition of CG6234 by RNA interference resulted in morphological defects in embryos, suggesting the involvement of this gene in germ band retraction. Conclusion Our data have yielded a list of developmentally regulated D. melanogaster genes and their expression profiles during embryogenesis and provide new information on the spatiotemporal expression patterns of several uncharacterized genes. In particular, we recovered a substantial number of unknown genes encoding putative secreted and transmembrane proteins, suggesting new components of signaling pathways that might be incorporated within the existing regulatory networks controlling D. melanogaster embryogenesis. These genes are also good candidates for additional targeted functional analyses similar to those we conducted for CG6234. See related minireview by Vichas and Zallen: PMID:19772636

  20. Pollen embryogenesis to induce, detect, and analyze mutants

    SciTech Connect

    Constantin, M.J.

    1981-01-01

    The development of fully differentiated plants from individual pollen grains through a series of developmental phases that resemble embryogenesis beginning with the zygote was demonstrated during the mid-1960's. This technology opened the door to the use of haploid plants (sporophytes with the gametic number of chromosomes) for plant breeding and genetic studies, biochemical and metabolic studies, and the selection of mutations. Although pollen embryogenesis has been demonstrated successfully in numerous plant genera, the procedure cannot as yet be used routinely to generate large populations of plants for experiments. Practical results from use of the technology in genetic toxicology research to detect mutations have failed to fully realize the theoretical potential; further developments of the technology could overcome the limitations. Pollen embryogenesis could be used to develop plants from mutant pollen grains to verify that genetic changes are involved. Through either spontaneous or induced chromosome doubling, these plants can be made homozygous and used to analyze genetically the mutants involved. The success of this approach will depend on the mutant frequency relative to the fraction of pollen grains that undergo embryogenesis; these two factors will dictate population size needed for success. Research effort is needed to further develop pollen embryogenesis for use in the detection of genotoxins under both laboratory and in situ conditions.

  1. Induction and characterization of oil palm (Elaeis guineensis Jacq.) pro-embryogenic masses.

    PubMed

    Pádua, Marlúcia S; Paiva, Luciano V; Labory, Claúdia R G; Alves, Eduardo; Stein, Vanessa C

    2013-01-01

    Oil palm is one of the most economically valuable oil seed plants, but the expansion of plantations has been limited by availability of seedlings, as the conventional propagation is through seeds, which have low germination rates. One possible solution for the large-scale production is the use of somatic embryogenesis. The aim of this study was evaluate the effects auxins 2,4-D and picloram on the induction of pro-embryogenic masses in E.guineenesis hybrid leaf explants and characterize, regarding embryogenic characteristics, with cytochemical and ultrastructural analysis. Specifically, in vitro plantlets leaves fragments were inoculated in Y3 culture medium supplemented by 2.4-D or picloram at different concentrations (0.0, 1.0, 3.0, 6.0 and 9.0 mg l⁻¹). After 90 days the presence/ absence of cell masses were evaluated. Both growth regulators efficiently induced cellular masses regardless of the concentrations applied. As the cell masses were not homogeneously formed, they were classified according to color and shape into four types: TYPE 1--elongated and translucent, TYPE 2--uneven and translucent, TYPE 3--globular and beige, TYPE 4--globular and white. Based on the anatomical and ultrastructural features, TYPE 2, 3 and 4 cell masses were considered to have the highest embryogenic potential and therefore may be most suited to large-scale vegetative propagation of oil palm. PMID:24270837

  2. Comparative expression analysis of multiple PDK genes in Xenopus laevis during oogenesis, maturation, fertilization, and early embryogenesis.

    PubMed

    Tokmakov, Alexander A; Terazawa, Yumiko; Ikeda, Mariko; Shirouzu, Mikako; Fukami, Yasuo; Yokoyama, Shigeyuki

    2009-03-01

    The complete family of expressed pyruvate dehydrogenase kinase (PDK) genes in the tissues of the African clawed frog, Xenopus laevis, consists of four members. Our previous study [Terazawa, Y., Tokmakov, A., Shirouzu, M., Yokoyama, S., 2005. Molecular cloning and expression analysis of PDK family genes in Xenopus laevis reveal oocyte-specific PDK isoform. Biochem. Biophys. Res. Commun. 338, 1798-1804] revealed that expression patterns of PDK genes differ greatly in the oocytes and somatic tissues of the adult frog. In the present work, using quantitative reverse-transcriptase PCR analysis, we demonstrate that the major transition from the oocyte-specific to somatic tissue-specific xPDK expression pattern occurs at the late stages of Xenopus embryogenesis after mid-blastula transition (MBT). Also, we show that the content of mRNA for xPDKo3, which is the predominant PDK isoform in oocytes and eggs, increases by about 3-fold during maturation. Other PDK family genes are down-regulated during oogenesis, thus being at their lowest expression levels in the grown-up oocytes, matured eggs, and early embryos. The expression of all PDK genes increases several-fold in the embryogenesis following MBT. Analysis of protein expression using an antibody raised against C-terminal of xPDKo3 confirmed isoform-specific up-regulation of xPDKo3 late in maturation and revealed cytoplasmic and mitochondrial localization of this protein. Bioinformatics and mass-spectrometric analyses allowed identification of an N-terminal mitochondrial targeting signal and a peptide cleavage site in xPDKo3 molecule. PMID:19084614

  3. Axes, planes and tubes, or the geometry of embryogenesis.

    PubMed

    Brauckmann, Sabine

    2011-12-01

    The paper presents selected figures of chick embryogenesis as depicted in the classic studies of Caspar Friedrich Wolff (1734-1794), Christian Heinrich Pander (1794-1865) and Karl Ernst von Baer (1792-1786). My main objective here is (1) to demonstrate how the imagery of Wolff, Pander and Baer attempted to project an image of a 3-dimensional rotating body into static figures on paper by means of linear contours, and (2) to ponder on the efficacy and pervasiveness of dots, lines and arrows for depicting embryogenesis. PMID:22035710

  4. Chimerism in humans after intragenic recombination at the haptoglobin locus during early embryogenesis.

    PubMed

    Asakawa, J; Kodaira, M; Nakamura, N; Satoh, C; Fujita, M

    1999-08-31

    The human haptoglobin (HP) HP*2 allele contains a 1.7-kilobase (kb) intragenic duplication that arose after a unique nonhomologous recombination between the prototype HP*1 alleles. During a genetic screening of 13,000 children of survivors exposed to atomic-bomb radiation and 10,000 children of unexposed persons, two children suspected of carrying de novo mutations at the haptoglobin locus were identified (one in each group). DNA analyses of single-cell-derived colonies of Epstein-Barr virus-transformed B cells revealed that the two children were mosaics comprising HP*2/HP*2 and HP*2/HP*1 cells at a ratio of approximately 3:1. We infer that the latter cells are caused by reversion of one HP*2 allele to HP*1 through an intramolecular homologous recombination between the duplicated segments of the Hp*2 allele that excised one of the segments. Because the mosaicism is substantial (approximately 25%), this recombination must have occurred in early embryogenesis. The frequency of finding these children and the extent of their mosaicisms corresponds to an HP*2 to HP*1 reversion rate of 8 x 10(-6) per cell during development. This leads to the prediction that the HP*1 allele also will be represented, although usually at a very low frequency, in any HP2-2 person. We tested this prediction by using PCR for a single individual and found the HP*1 allele at frequencies of 4 x 10(-6) and 3 x 10(-6) in somatic and sperm cells. The HP*1 allele was detected by PCR in all four other HP2-2 individuals, which supports the regular but rare occurrence somatically of homologous recombination within duplicated regions in humans, in agreement with previous observations in mouse and Drosophila. PMID:10468605

  5. Characterization of conservative somatic instability of the CAG repeat region in Huntington`s disease

    SciTech Connect

    Schaefer, F.V.; Calikoglu, A.S.; Whetsell, L.H.

    1994-09-01

    Instability and enlargement of a CAG repeat region at the beginning of the huntingtin gene (IT-15) has been linked with Huntington`s disease. The CAG repeat size shows a highly significant correlation with age-of-onset of clinicial features in individuals with 40 or more repeats who have Huntington disease. The clinical status of nonsymptomatic individuals with 30 to 39 CAG repeats is considered ambiguous. In order to define more carefully the nature of the HD expansion instability, we examined patients in our HD population using a discriminating fluorescence-based PCR approach. The degree of somatic mutation increases with both earlier age of onset and the size of the inherited allele. A single prominent band one repeat larger than the index peak was typical in individuals with 40-41 CAG repeats. Three to four larger bands are typically discerned in individuals with 50 or more repeats. In an extreme example, an individual with approximately 95 repeats had at least 8 prominent bands. Plotting the degree of somatic mutation relative to the size of the HD allele shows somatic mutation activity increases with size. By this approach 40-60% of the alleles in a 40-41 CAG repeat HD loci is represented in the primary allele. In contrast, the primary allele represents a relatively minor proportion of the total alleles for expansions greater than 50 CAG repeats (10-20%). The limited range of somatic mutation suggest that the instability is restricted to very early stages of embryogenesis before tissue development diverges or that persistent somatic instability occurs at a slow rate. Therefore, the properties of somatic instability in Huntington`s disease have aspects that are both in common but also different from that found in other trinucleotide repeat expanding diseases such as myotonic muscular dystrophy and fragile X syndrome.

  6. Globin gene expression in somatic cell hybrids.

    PubMed

    Anderson, W F; Chiang, Y L; Sanders-Haigh, L; Ley, T J

    1983-01-01

    Fusions between somatic cell lines have previously yielded evidence for the existence of trans-acting gene regulatory factors. For this reason, we developed a cell line containing a "locked in" human 11-X translocation chromosome (containing the beta-globin-like gene cluster) in MEL cells. The human 11-X chromosome is stably integrated in the "M11-X" cell line, and single-copy human gamma and beta genes are present. After induction with HMBA, M11-X cells produced 500 copies per cell of correctly initiated, processed, and terminated human beta-globin mRNA; authentic human beta-globin chains were also produced at a low level. Despite the presence of normally arranged human gamma-globin genes, no gamma-globin mRNA could be detected after HMBA induction. However, cytosine residues near the gamma-globin gene promoters are completely methylated in these cells, suggesting that the gamma-globin genes may be repressed in part by DNA methylation. The pattern of human globin gene expression in M11-X cells may be affected by methylation and/or by trans-acting factors produced by these tetraploid cells. PMID:6320217

  7. Are Early Somatic Embryos of the Norway Spruce (Picea abies (L.) Karst.) Organised?

    PubMed Central

    Petrek, Jiri; Zitka, Ondrej; Adam, Vojtech; Bartusek, Karel; Anjum, Naser A.; Pereira, Eduarda; Havel, Ladislav; Kizek, Rene

    2015-01-01

    Background Somatic embryogenesis in conifer species has great potential for the forestry industry. Hence, a number of methods have been developed for their efficient and rapid propagation through somatic embryogenesis. Although information is available regarding the previous process-mediated generation of embryogenic cells to form somatic embryos, there is a dearth of information in the literature on the detailed structure of these clusters. Methodology/Principal Findings The main aim of this study was to provide a more detailed structure of the embryogenic tissue clusters obtained through the in vitro propagation of the Norway spruce (Picea abies (L.) Karst.). We primarily focused on the growth of early somatic embryos (ESEs). The data on ESE growth suggested that there may be clear distinctions between their inner and outer regions. Therefore, we selected ESEs collected on the 56th day after sub-cultivation to dissect the homogeneity of the ESE clusters. Two colourimetric assays (acetocarmine and fluorescein diacetate/propidium iodide staining) and one metabolic assay based on the use of 2,3,5-triphenyltetrazolium chloride uncovered large differences in the metabolic activity inside the cluster. Next, we performed nuclear magnetic resonance measurements. The ESE cluster seemed to be compactly aggregated during the first four weeks of cultivation; thereafter, the difference between the 1H nuclei concentration in the inner and outer clusters was more evident. There were clear differences in the visual appearance of embryos from the outer and inner regions. Finally, a cluster was divided into six parts (three each from the inner and the outer regions of the embryo) to determine their growth and viability. The innermost embryos (centripetally towards the cluster centre) could grow after sub-cultivation but exhibited the slowest rate and required the longest time to reach the common growth rate. To confirm our hypothesis on the organisation of the ESE cluster, we investigated the effect of cluster orientation on the cultivation medium and the influence of the change of the cluster’s three-dimensional orientation on its development. Maintaining the same position when transferring ESEs into new cultivation medium seemed to be necessary because changes in the orientation significantly affected ESE growth. Conclusions and Significance This work illustrated the possible inner organisation of ESEs. The outer layer of ESEs is formed by individual somatic embryos with high metabolic activity (and with high demands for nutrients, oxygen and water), while an embryonal group is directed outside of the ESE cluster. Somatic embryos with depressed metabolic activity were localised in the inner regions, where these embryonic tissues probably have a very important transport function. PMID:26624287

  8. Drosophila Embryogenesis Scales Uniformly across Temperature in Developmentally Diverse Species

    PubMed Central

    Kuntz, Steven G.; Eisen, Michael B.

    2014-01-01

    Temperature affects both the timing and outcome of animal development, but the detailed effects of temperature on the progress of early development have been poorly characterized. To determine the impact of temperature on the order and timing of events during Drosophila melanogaster embryogenesis, we used time-lapse imaging to track the progress of embryos from shortly after egg laying through hatching at seven precisely maintained temperatures between 17.5°C and 32.5°C. We employed a combination of automated and manual annotation to determine when 36 milestones occurred in each embryo. D. melanogaster embryogenesis takes 33 hours at 17.5°C, and accelerates with increasing temperature to a low of 16 hours at 27.5°C, above which embryogenesis slows slightly. Remarkably, while the total time of embryogenesis varies over two fold, the relative timing of events from cellularization through hatching is constant across temperatures. To further explore the relationship between temperature and embryogenesis, we expanded our analysis to cover ten additional Drosophila species of varying climatic origins. Six of these species, like D. melanogaster, are of tropical origin, and embryogenesis time at different temperatures was similar for them all. D. mojavensis, a sub-tropical fly, develops slower than the tropical species at lower temperatures, while D. virilis, a temperate fly, exhibits slower development at all temperatures. The alpine sister species D. persimilis and D. pseudoobscura develop as rapidly as tropical flies at cooler temperatures, but exhibit diminished acceleration above 22.5°C and have drastically slowed development by 30°C. Despite ranging from 13 hours for D. erecta at 30°C to 46 hours for D. virilis at 17.5°C, the relative timing of events from cellularization through hatching is constant across all species and temperatures examined here, suggesting the existence of a previously unrecognized timer controlling the progress of embryogenesis that has been tuned by natural selection as each species diverges. PMID:24762628

  9. NO, ROS, and cell death associated with caspase-like activity increase in stress-induced microspore embryogenesis of barley.

    PubMed

    Rodríguez-Serrano, María; Bárány, Ivett; Prem, Deepak; Coronado, María-José; Risueño, María C; Testillano, Pilar S

    2012-03-01

    Under specific stress treatments (cold, starvation), in vitro microspores can be induced to deviate from their gametophytic development and switch to embryogenesis, forming haploid embryos and homozygous breeding lines in a short period of time. The inductive stress produces reactive oxygen species (ROS) and nitric oxide (NO), signalling molecules mediating cellular responses, and cell death, modifying the embryogenic microspore response and therefore, the efficiency of the process. This work analysed cell death, caspase 3-like activity, and ROS and NO production (using fluorescence probes and confocal analysis) after inductive stress in barley microspore cultures and embryogenic suspension cultures, as an in vitro system which permitted easy handling for comparison. There was an increase in caspase 3-like activity and cell death after stress treatment in microspore and suspension cultures, while ROS increased in non-induced microspores and suspension cultures. Treatments of the cultures with a caspase 3 inhibitor, DEVD-CHO, significantly reduced the cell death percentages. Stress-treated embryogenic suspension cultures exhibited high NO signals and cell death, while treatment with S-nitrosoglutathione (NO donor) in control suspension cultures resulted in even higher cell death. In contrast, in microspore cultures, NO production was detected after stress, and, in the case of 4-day microspore cultures, in embryogenic microspores accompanying the initiation of cell divisions. Subsequent treatments of stress-treated microspore cultures with ROS and NO scavengers resulted in a decreasing cell death during the early stages, but later they produced a delay in embryo development as well as a decrease in the percentage of embryogenesis in microspores. Results showed that the ROS increase was involved in the stress-induced programmed cell death occurring at early stages in both non-induced microspores and embryogenic suspension cultures; whereas NO played a dual role after stress in the two in vitro systems, one involved in programmed cell death in embryogenic suspension cultures and the other in the initiation of cell division leading to embryogenesis in reprogrammed microspores. PMID:22197894

  10. NO, ROS, and cell death associated with caspase-like activity increase in stress-induced microspore embryogenesis of barley

    PubMed Central

    Rodríguez-Serrano, María; Bárány, Ivett; Prem, Deepak; Coronado, María-José; Risueño, María C.; Testillano, Pilar S.

    2012-01-01

    Under specific stress treatments (cold, starvation), in vitro microspores can be induced to deviate from their gametophytic development and switch to embryogenesis, forming haploid embryos and homozygous breeding lines in a short period of time. The inductive stress produces reactive oxygen species (ROS) and nitric oxide (NO), signalling molecules mediating cellular responses, and cell death, modifying the embryogenic microspore response and therefore, the efficiency of the process. This work analysed cell death, caspase 3-like activity, and ROS and NO production (using fluorescence probes and confocal analysis) after inductive stress in barley microspore cultures and embryogenic suspension cultures, as an in vitro system which permitted easy handling for comparison. There was an increase in caspase 3-like activity and cell death after stress treatment in microspore and suspension cultures, while ROS increased in non-induced microspores and suspension cultures. Treatments of the cultures with a caspase 3 inhibitor, DEVD-CHO, significantly reduced the cell death percentages. Stress-treated embryogenic suspension cultures exhibited high NO signals and cell death, while treatment with S-nitrosoglutathione (NO donor) in control suspension cultures resulted in even higher cell death. In contrast, in microspore cultures, NO production was detected after stress, and, in the case of 4-day microspore cultures, in embryogenic microspores accompanying the initiation of cell divisions. Subsequent treatments of stress-treated microspore cultures with ROS and NO scavengers resulted in a decreasing cell death during the early stages, but later they produced a delay in embryo development as well as a decrease in the percentage of embryogenesis in microspores. Results showed that the ROS increase was involved in the stress-induced programmed cell death occurring at early stages in both non-induced microspores and embryogenic suspension cultures; whereas NO played a dual role after stress in the two in vitro systems, one involved in programmed cell death in embryogenic suspension cultures and the other in the initiation of cell division leading to embryogenesis in reprogrammed microspores. PMID:22197894

  11. Genotoxic effects of cisplatin in somatic tissue of Drosophila melanogaster

    SciTech Connect

    Katz, A.J.

    1987-01-01

    Third instar larvae of Drosophila melanogaster transdihybrid for mwh and flr were exposed to varying concentrations of cisplatin by feeding on dry media wetted with aqueous solutions of the test compound. Larval feeding continued until pupation, and surviving transdihybrid adults were collected seven days following commencement of feeding. Wings of adults were removed and scored under 400X magnification for the presence of twin spots and single spots comprised of clones of cells possessing malformed wing hairs. Cisplatin was found to induce both twin spots and single spots, and significant linear concentration-response relationships were obtained with respect to the induction of all endpoints. This capacity to induce mitotic exchange in the somatic tissue of Drosophila compares well with the compound's reported ability to induce chromosome breaks in Drosophila germ cells. However, not all compounds possess similar genotoxic profiles in the somatic an germ tissue of Drosophila.

  12. D-MEF2: a MADS box transcription factor expressed in differentiating mesoderm and muscle cell lineages during Drosophila embryogenesis.

    PubMed

    Lilly, B; Galewsky, S; Firulli, A B; Schulz, R A; Olson, E N

    1994-06-01

    The myocyte enhancer factor (MEF) 2 family of transcription factors has been implicated in the regulation of muscle transcription in vertebrates. We have cloned a protein from Drosophila, termed D-MEF2, that shares extensive amino acid homology with the MADS (MCM1, Agamous, Deficiens, and serum-response factor) domains of the vertebrate MEF2 proteins. D-mef2 gene expression is first detected during Drosophila embryogenesis within mesodermal precursor cells prior to specification of the somatic and visceral muscle lineages. Expression of D-mef2 is dependent on the mesodermal determinants twist and snail but independent of the homeobox-containing gene tinman, which is required for visceral muscle and heart development. D-mef2 expression precedes that of the MyoD homologue, nautilus, and, in contrast to nautilus, D-mef2 appears to be expressed in all somatic and visceral muscle cell precursors. Its temporal and spatial expression patterns suggest that D-mef2 may play an important role in commitment of mesoderm to myogenic lineages. PMID:8202544

  13. GENOMIC ANALYSIS OF CANCER TISSUE REVEALS THAT SOMATIC MUTATIONS COMMONLY OCCUR IN A SPECIFIC MOTIF

    PubMed Central

    Makridakis, Nick M; Ferraz, Lúcio Fábio Caldas; Reichardt, Juergen KV

    2009-01-01

    Somatic mutations are hallmarks of cancer progression. We sequenced 26 matched human prostate tumor and constitutional DNA samples for somatic alterations in the SRD5A2, HPRT, and HSD3B2 genes, and identified 71 nucleotide substitutions. 79% (56/71) of these substitutions occur within a WKVnRRRnVWK sequence (THEMIS motif; W= A/T, K= G/T, V= G/A/C, R= purine (A/G) and n= any nucleotide), with one mismatch allowed. Literature searches identified this motif with one mismatch allowed in 66% (37/ 56) of the somatic prostate cancer mutations and in 74% (90/ 122) of the somatic breast cancer mutations found in all human genes analyzed. We also found the THEMIS motif with one allowed mismatch in 88% (23/26) of the ras1 gene somatic mutations formed in the SENCAR (SENsitive to skin CARcinogenesis) mouse model, after induction of error-prone DNA repair following mutagenic treatment. The high prevalence of the motif in each of the above mentioned cases cannot be explained by chance (p < 0.046). We further identified 27 somatic mutations in the error-prone DNA polymerase genes pol η, pol κ and pol β in these prostate cancer patients. The data suggest that most somatic nucleotide substitutions in human cancer may occur in sites that conform to the THEMIS motif. These mutations may be caused by “mutator” mutations in error-prone DNA polymerase genes. PMID:18623241

  14. Somatic mutations in cancer development

    PubMed Central

    2011-01-01

    The transformation of a normal cell into a cancer cell takes place through a sequence of a small number of discrete genetic events, somatic mutations: thus, cancer can be regarded properly as a genetic disease of somatic cells. The analogy between evolution of organisms and evolution of cell populations is compelling: in both cases what drives change is mutation, but it is Darwinian selection that enables clones that have a growth advantage to expand, thus providing a larger target size for the next mutation to hit. The search for molecular lesions in tumors has taken on a new dimension thanks to two powerful technologies: the micro-arrays for quantitative analysis of global gene expresssion (the transcriptome); and ‘deep’ sequencing for the global analysis of the entire genome (or at least the exome). The former offers the most complete phenotypic characterization of a tumor we could ever hope for – we could call this the ultimate phenotype; the latter can identify all the somatic mutations in an individual tumor – we could call this the somatic genotype. However, there is definitely the risk that while we are ‘drowned by data, we remain thirsty for knowledge’. If we want to heed the teachings of Lorenzo Tomatis, I think the message is clear: we ought to take advantage of the new powerful technologies – not by becoming their slaves, but remaining their masters. Identifying somatic mutations in a tumor is important not because it qualifies for ‘oncogenomics’, but because through a deeper understanding of the nature of that particular tumor it can help us to optimize therapy or to design new therapeutic approaches. PMID:21489208

  15. Somatic mutations in cancer development.

    PubMed

    Luzzatto, Lucio

    2011-01-01

    The transformation of a normal cell into a cancer cell takes place through a sequence of a small number of discrete genetic events, somatic mutations: thus, cancer can be regarded properly as a genetic disease of somatic cells. The analogy between evolution of organisms and evolution of cell populations is compelling: in both cases what drives change is mutation, but it is Darwinian selection that enables clones that have a growth advantage to expand, thus providing a larger target size for the next mutation to hit. The search for molecular lesions in tumors has taken on a new dimension thanks to two powerful technologies: the micro-arrays for quantitative analysis of global gene expresssion (the transcriptome); and 'deep' sequencing for the global analysis of the entire genome (or at least the exome). The former offers the most complete phenotypic characterization of a tumor we could ever hope for--we could call this the ultimate phenotype; the latter can identify all the somatic mutations in an individual tumor--we could call this the somatic genotype. However, there is definitely the risk that while we are 'drowned by data, we remain thirsty for knowledge'. If we want to heed the teachings of Lorenzo Tomatis, I think the message is clear: we ought to take advantage of the new powerful technologies--not by becoming their slaves, but remaining their masters. Identifying somatic mutations in a tumor is important not because it qualifies for 'oncogenomics', but because through a deeper understanding of the nature of that particular tumor it can help us to optimize therapy or to design new therapeutic approaches. PMID:21489208

  16. Expression of the Hsp23 chaperone during Drosophila embryogenesis: association to distinct neural and glial lineages

    PubMed Central

    Michaud, Sébastien; Tanguay, Robert M

    2003-01-01

    Background In addition to their strong induction following stress, small heat shock proteins (Hsp) are also expressed during development in a wide variety of organisms. However, the precise identity of cell(s) expressing these proteins and the functional contribution of small heat shock proteins in such developmental context remain to be determined. The present study provides a detailed description of the Drosophila small heat shock protein Hsp23 expression pattern during embryogenesis and evaluates its functional contribution to central nervous system development. Results Throughout embryogenesis, Hsp23 is expressed in a stage-specific manner by a restricted number of neuronal and glial lineages of the central nervous system. Hsp23 is also detected in the amnioserosa and within a single lateral chordotonal organ. Its expression within the MP2 lineage does not require the presence of a functional midline nor the activity of the Notch signaling pathway. Transactivation assays demonstrate that transcription factors implicated in the differentiation of the midline also regulate hsp23 promoter activity. Phenotypic analysis of a transgenic line exhibiting loss of Hsp23 expression in the central nervous system suggests that Hsp23 is not required for development and function of this tissue. Likewise, its overexpression does not cause deleterious effects, as development remains unaffected. Conclusions Based on the presented data, we suggest that the tightly regulated developmental expression of Hsp23 is not actively involved in cell differentiation and central nervous system development per se but rather reflects a putative role in preventive "pre-stress" neuroprotection or in non-vital process(es) common to the identified cell lineages. PMID:14617383

  17. Detection of somatic mosaicism in DMD using computer-assisted laser densitometry

    SciTech Connect

    Sutherland, J.E.; Allingham-Hawkins, D.J.; MacKenzie, J.

    1994-09-01

    Approximately two-thirds of Duchenne muscular dystrophy (DMD) patients have a deletion in the dystrophin gene located at Xp21.1. Two PCR-based multiplex systems have been developed which detect 98% of deletions in affected males. Diagnosis of carrier females requires densitometry of PCR products following gel electrophoresis to calculate dosage of specific exons. We have developed a system in which fluorescently labelled PCR products are analysed using a GENESCANNER automated fragment analyser (ABI). Dosage is determined using computer-assisted laser densitometry (CALD). Recently, we diagnosed somatic mosaicism in the mother of an affected boy using this method. PCR analysis showed that the patient had a deletion that included exons 47-51 of his dystrophin gene. CALD analysis on the patient`s 36-year-old mother revealed a 29-34% reduction in the intensity of the bands corresponding to the deleted region of the gene rather than the 50% reduction normally seen in carrier females. A skin biopsy was obtain and monoclonal fibroblast colonies were tested by CALD for the deletion. Four of the twenty colonies screened were found to be deleted while the remaining colonies had two intact copies of the gene. We conclude that this patient is a somatic mosaic for DMD and that the mutation was the result of a post-zygotic event. This is the only case of somatic mosaicism detected among 800 women from 400 DMD families tested using CALD in our laboratory. At least one other case of possible somatic mosaicism has been reported but not confirmed. Germinal mosaicism is thought to occur in approximately 10% of mothers of sporadic DMD patients. Our findings indicate that somatic mosaicism is a much rarer condition among DMD carriers, thus suggesting that mitotic mutations in the dystrophin gene are more likely to occur later in embryogenesis after differentiation of the germline.

  18. Somatic mutation, genomic variation, and neurological disease.

    PubMed

    Poduri, Annapurna; Evrony, Gilad D; Cai, Xuyu; Walsh, Christopher A

    2013-07-01

    Genetic mutations causing human disease are conventionally thought to be inherited through the germ line from one's parents and present in all somatic (body) cells, except for most cancer mutations, which arise somatically. Increasingly, somatic mutations are being identified in diseases other than cancer, including neurodevelopmental diseases. Somatic mutations can arise during the course of prenatal brain development and cause neurological disease-even when present at low levels of mosaicism, for example-resulting in brain malformations associated with epilepsy and intellectual disability. Novel, highly sensitive technologies will allow more accurate evaluation of somatic mutations in neurodevelopmental disorders and during normal brain development. PMID:23828942

  19. Somatic Mutation, Genomic Variation, and Neurological Disease

    PubMed Central

    Poduri, Annapurna; Evrony, Gilad D.; Cai, Xuyu; Walsh, Christopher A.

    2014-01-01

    Genetic mutations causing human disease are conventionally thought to be inherited through the germ line from one’s parents and present in all somatic (body) cells, except for most cancer mutations, which arise somatically. Increasingly, somatic mutations are being identified in diseases other than cancer, including neurodevelopmental diseases. Somatic mutations can arise during the course of prenatal brain development and cause neurological disease—even when present at low levels of mosaicism, for example—resulting in brain malformations associated with epilepsy and intellectual disability. Novel, highly sensitive technologies will allow more accurate evaluation of somatic mutations in neurodevelopmental disorders and during normal brain development. PMID:23828942

  20. Xenopus sonic hedgehog as a potential morphogen during embryogenesis and thyroid hormone-dependent metamorphosis.

    PubMed Central

    Stolow, M A; Shi, Y B

    1995-01-01

    The hedgehog family of proteins have been implicated as important signaling molecules in establishing cell positional information and tissue patterning. Here we present the cloning and characterization of a hedgehog homologue from Xenopus laevis similar to the sonic class of vertebrate hedgehog genes. We isolated Xenopus hedgehog (Xhh) from a subtractive hybridization screen designed to identify genes induced by thyroid hormone during metamorphosis of the X.laevis gastrointestinal tract. In the intestine, Xhh mRNA expression was up-regulated at the climax of metamorphosis (stage 62) when intestinal epithelium underwent morphogenesis. Treatment of pre-metamorphic tadpoles with exogenous thyroid hormone (TH) resulted in a similar pattern of Xhh induction. Furthermore, TH induction was resistant to inhibitors of protein synthesis suggesting that Xhh is a direct thyroid hormone response gene. The expression and TH regulation of Xhh was not limited to the intestine, but was also observed in the limb and a mixture of pancreas and stomach. Throughout development, Xhh mRNA was present at varying levels with the earliest expression being detected at neurula stage. The highest levels of Xhh were observed between stages 33 and 40 shortly before tadpole feeding begins. Whole mount in situ hybridization analysis of Xhh expression in pre-hatching, stage 32 tadpoles demonstrated staining in the notochord and floor plate similar to that observed for other vertebrate hedgehog genes. Together, these data suggest a putative role for Xhh in organ development during both amphibian embryogenesis and metamorphosis. Images PMID:7630736

  1. Morphogenesis of the somatic musculature in Drosophila melanogaster.

    PubMed

    Schulman, Victoria K; Dobi, Krista C; Baylies, Mary K

    2015-01-01

    In Drosophila melanogaster, the somatic muscle system is first formed during embryogenesis, giving rise to the larval musculature. Later during metamorphosis, this system is destroyed and replaced by an entirely new set of muscles in the adult fly. Proper formation of the larval and adult muscles is critical for basic survival functions such as hatching and crawling (in the larva), walking and flying (in the adult), and feeding (at both larval and adult stages). Myogenesis, from mononucleated muscle precursor cells to multinucleated functional muscles, is driven by a number of cellular processes that have begun to be mechanistically defined. Once the mesodermal cells destined for the myogenic lineage have been specified, individual myoblasts fuse together iteratively to form syncytial myofibers. Combining cytoplasmic contents demands a level of intracellular reorganization that, most notably, leads to redistribution of the myonuclei to maximize internuclear distance. Signaling from extending myofibers induces terminal tendon cell differentiation in the ectoderm, which results in secure muscle-tendon attachments that are critical for muscle contraction. Simultaneously, muscles become innervated and undergo sarcomerogenesis to establish the contractile apparatus that will facilitate movement. The cellular mechanisms governing these morphogenetic events share numerous parallels to mammalian development, and the basic unit of all muscle, the myofiber, is conserved from flies to mammals. Thus, studies of Drosophila myogenesis and comparisons to muscle development in other systems highlight conserved regulatory programs of biomedical relevance to general muscle biology and studies of muscle disease. PMID:25758712

  2. Morphogenesis of the somatic musculature in Drosophila melanogaster

    PubMed Central

    Schulman, Victoria K.; Dobi, Krista C.; Baylies, Mary K.

    2015-01-01

    In Drosophila melanogaster, the somatic muscle system is first formed during embryogenesis, giving rise to the larval musculature. Later during metamorphosis, this system is destroyed and replaced by an entirely new set of muscles in the adult fly. Proper formation of the larval and adult muscles is critical for basic survival functions such as hatching and crawling (in the larva), walking and flying (in the adult), and feeding (at both larval and adult stages). Myogenesis, from mononucleated muscle precursor cells to multinucleated functional muscles, is driven by a number of cellular processes that have begun to be mechanistically defined. Once themesodermal cells destined for themyogenic lineage have been specified, individual myoblasts fuse together iteratively to form syncytial myofibers. Combining cytoplasmic contents demands a level of intracellular reorganization that, most notably, leads to redistribution of the myonuclei to maximize internuclear distance. Signaling from extending myofibers induces terminal tendon cell differentiation in the ectoderm, which results in secure muscle-tendon attachments that are critical formuscle contraction. Simultaneously, muscles become innervated and undergo sarcomerogenesis to establish the contractile apparatus that will facilitate movement. The cellular mechanisms governing these morphogenetic events share numerous parallels to mammalian development, and the basic unit of all muscle, the myofiber, is conserved from flies to mammals. Thus, studies of Drosophila myogenesis and comparisons to muscle development in other systems highlight conserved regulatory programs of biomedical relevance to general muscle biology and studies of muscle disease. PMID:25758712

  3. Cracking the egg: virtual embryogenesis of real robots.

    PubMed

    Cussat-Blanc, Sylvain; Pollack, Jordan

    2014-01-01

    All multicellular living beings are created from a single cell. A developmental process, called embryogenesis, takes this first fertilized cell down a complex path of reproduction, migration, and specialization into a complex organism adapted to its environment. In most cases, the first steps of the embryogenesis take place in a protected environment such as in an egg or in utero. Starting from this observation, we propose a new approach to the generation of real robots, strongly inspired by living systems. Our robots are composed of tens of specialized cells, grown from a single cell using a bio-inspired virtual developmental process. Virtual cells, controlled by gene regulatory networks, divide, migrate, and specialize to produce the robot's body plan (morphology), and then the robot is manually built from this plan. Because the robot is as easy to assemble as Lego, the building process could be easily automated. PMID:24730763

  4. Chronic functional somatic symptoms: a single syndrome?

    PubMed Central

    olde Hartman, Tim C; Lucassen, Peter L B J; van de Lisdonk, Eloy H; Bor, Hans H J; van Weel, Chris

    2004-01-01

    Background: Reliable longitudinal data of patients with functional somatic symptoms in general practice are lacking. Aims: To identify distinctive features in patients with chronic functional somatic symptoms, and to determine whether these symptoms support the hypothesis of the existence of specific somatic syndromes. Design of study: Observational study, with a comparison control group. Setting: Four primary care practices affiliated with the University of Nijmegen in the Netherlands. Method: One hundred and eighty-two patients diagnosed between 1998 and 2002 as having chronic functional somatic symptoms and 182 controls matched by age, sex, socioeconomic status, and practice were included. Data on comorbidity, referrals, diagnostic tests, and hospital admissions over a period of 10 years prior to the diagnosis were collected. Medication use and number of visits to the general practitioner (GP) were extracted from the moment computerised registration was started. Results: In the 10 years before the diagnosis of chronic functional somatic symptoms, significantly more patients than controls presented functional somatic symptoms in at least two body systems, and used more somatic and psychotropic drugs. They visited the GP twice as much, statistically had significantly more psychiatric morbidity, and were referred more often to mental health workers and somatic specialists. The number of patients undergoing diagnostic tests was higher for patients with chronic functional somatic symptoms than for controls, but hospital admissions rates were equal. Conclusion: Patients with chronic functional somatic symptoms have a great diversity of functional somatic symptoms. They use more somatic and psychotropic drugs than controls in the years before diagnosis. Moreover, they show high rates of referrals and psychiatric morbidity. The diversity of symptoms of patients with chronic functional somatic symptoms supports the concept that symptoms do not cluster in well defined distinct syndromes. Therefore, patients with chronic functional somatic symptoms should preferably not be classified into medical subspecialty syndromes. PMID:15588538

  5. Transcription of subtelomere tandemly repetitive DNA in chicken embryogenesis.

    PubMed

    Trofimova, Irina; Chervyakova, Darya; Krasikova, Alla

    2015-09-01

    Transcription of tandemly repetitive DNA in embryogenesis seems to be of special interest due to a crucial role of non-coding RNAs in many aspects of development. However, only a few data are available on tandem repeats transcription at subtelomere regions of chromosomes during vertebrate embryogenesis. To reduce this gap, we examined stage and tissue-specific pattern of subtelomeric PO41 (pattern of 41 bp) tandem repeat transcription during embryogenesis of chicken (Gallus gallus domesticus). Using whole-mount RNA fluorescent in situ hybridization and reverse transcription PCR with specific primers, we demonstrated that both strands of PO41 repeat are transcribed at each of the studied stages of chicken embryo development: from 7-8 HH to 20 HH stages. Subtelomere-derived transcripts localize in the nuclei of all cell types and throughout the all embryonic bodies: head, somites, tail, wings and buds. In embryo-dividing cells and cultured embryonic fibroblasts, PO41 RNAs envelop terminal regions of chromosomes. PO41-containing RNAs are predominantly single-stranded and can be polyadenylated, indicating appearance of non-nascent form of subtelomeric transcripts. PO41 repeat RNAs represent a rare example of ubiquitously transcribed non-coding RNAs, such as Xist/XIST RNA or telomere repeat-containing RNA. Distribution of PO41 repeat transcripts at different stages of embryo development and among cell types has extremely uniform pattern, indicating on possible universal functions of PO41 non-coding RNAs. PMID:26363798

  6. Global transcriptome analysis identifies regulated transcripts and pathways activated during oogenesis and early embryogenesis in atlantic cod

    PubMed Central

    Kleppe, Lene; Edvardsen, Rolf Brudvik; Furmanek, Tomasz; Taranger, Geir Lasse; Wargelius, Anna

    2014-01-01

    The molecular mechanisms underlying oogenesis and maternally controlled embryogenesis in fish are not fully understood, especially in marine species. Our aim was to study the egg and embryo transcriptome during oogenesis and early embryogenesis in Atlantic cod. Follicles from oogenesis stages (pre-, early-, and late-vitellogenic), ovulated eggs, and two embryonic stages (blastula, gastrula) were collected from broodstock fish and fertilized eggs. Gene expression profiles were measured in a 44 K oligo microarray consisting of 23,000 cod genes. Hundreds of differentially expressed genes (DEGs) were identified in the follicle stages investigated, implicating a continuous accumulation and degradation of polyadenylated transcripts throughout oogenesis. Very few DEGs were identified from ovulated egg to blastula, showing a more stable maternal RNA pool in early embryonic stages. The highest induction of expression was observed between blastula and gastrula, signifying the onset of zygotic transcription. During early vitellogenesis, several of the most upregulated genes are linked to nervous system signaling, suggesting increasing requirements for ovarian synaptic signaling to stimulate the rapid growth of oocytes. Highly upregulated genes during late vitellogenesis are linked to protein processing, fat metabolism, osmoregulation, and arrested meiosis. One of the genes with the highest upregulation in the ovulated egg is involved in oxidative phosphorylation, reflecting increased energy requirements during fertilization and the first rapid cell divisions of early embryogenesis. In conclusion, this study provides a large-scale presentation of the Atlantic cod's maternally controlled transcriptome in ovarian follicles through oogenesis, ovulated eggs, and early embryos. Mol. Reprod. Dev. 81: 619–635, 2014. © 2014 Wiley Periodicals, Inc. PMID:24687555

  7. The effects of Ostertagia occidentalis somatic antigens on ovine TLR2 and TLR4 expression

    PubMed Central

    BORJI, Hassan; HAGHPARAST, Alireza; SOLEIMANI, Nooshinmehr; AZIZZADEH, Mohammad; NAZEMSHIRAZI, Mohammad Hossein

    2015-01-01

    Background: Recognition of helminth-derived pathogen associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), including toll like receptors (TLRs) is the first step towards initiating anti–helminth immune responses. Methods : Using somatic antigens of Ostertagia occidentalis, an important abomasal parasite of ruminants, the expression of ovine TLR2 and TLR4 in peripheral blood mononuclear cells (PBMCs) was analyzed by real-time quatitative reverse-transcription polymerase chain reaction (qRT-PCR). Somatic antigens of O. occidentalis were prepared to stimulate ovine PBMCs in a time and dose dependent manner. Results : A high expression of TLR2 and TLR4 was observed in PBMCs cultured with somatic antigens of the parasites specially when PBMCs were cultured with 100 µg/ml of somatic antigens and incubated for 2h. Up-regulation of TLR2 expression was more pronounced and evident in our study. Conclsusion : Somatic antigens of O. occidentalis have immunostimulatory and dominant role on peripheral immune cells. This study provide for the first time evidence of induction of TLRs in ovine PBMCs by somatic antigen of O. occidentalis PMID:26622306

  8. Attachment in romantic relationships and somatization.

    PubMed

    Neumann, Eva; Sattel, Heribert; Gündel, Harald; Henningsen, Peter; Kruse, Johannes

    2015-02-01

    Adult attachment representations have been considered to play a role in the development and treatment of somatizing behavior. In this study, the associations between the two attachment dimensions avoidance and anxiety and dimensions of psychopathology (somatization, depression, and general anxiety) were explored. The sample consists of 202 outpatients diagnosed with a somatoform disorder. Data were collected via self-report measures. A path analysis shows that the two attachment dimensions are not directly associated with somatization. There are, however, significant indirect associations between attachment and somatization mediated by depression and general anxiety, which are more pronounced for attachment anxiety than for attachment avoidance. The findings reveal that a low level of attachment security in romantic relationships, especially an anxious stance toward the partner, comes along with poor mental health, which in turn is related to a preoccupation with somatic complaints. Implications for the treatment of somatizing patients are discussed. PMID:25594785

  9. Fertile fruit trees obtained by somatic hybridization: navel orange (Citrus sinensis) and Troyer citrange (C. sinensis x Poncirus trifoliata).

    PubMed

    Ohgawara, T; Kobayashi, S; Ishii, S; Yoshinaga, K; Oiyama, I

    1991-02-01

    Nucellar cell suspension protoplasts of navel orange (Citrus sinsensis Osb.) were chemically fused with mesophyll protoplasts of Troyer citrange (C. sinensis x Poncirus trifoliata) and cultured in hormone-free Murashige and Tucker medium containing 0.6 M sucrose. Two types of plant were regenerated through embryogenesis. One type showed intermediate mono-and difoliate leaves and the other types was identical to Troyer citrange. The regenerated plants with intermediate morphology were demonstrated by chromosome counts and rDNA analysis to be amphidiploid somatic hybrids. Five clones of these somatic hybrids were grafted in the field. After 4 years, they set flowers having a morphology intermediate between those of the two parents. The pollen grains showed high stainability and sufficient germinability, and were larger than those of Troyer citrange. The fruits of the somatic hybrids were large and spherical with thick rinds. Most of them contained seeds with normal germinability. These results indicate that somatic hybridization is a useful tool for Citrus breeding. PMID:24221194

  10. [Anatomic and morphological studies on the initiation of somatic embryos obtained from meristematic apexes of Musa sp.

    PubMed

    Vidal, M C; Vargas, T E; de García, E

    2000-01-01

    The origin of somatic embryos obtained from meristematic apexes of the Musa (AAA) clone "Gran enano" was analyzed through histological and morphological studies during the various development phases of the process. The research point out that somatic embryos developed directly from perivascular parenchyma cells of the leaves. Histological sections of globular embryos showed a radial disposition to cell and the existence of an epidermal layer that surrounds the embryo completely. When citocinine (Z or BA) was added, some embryos remained in globular stage with mild signs of enlargement but with no later development of invagination. Other's embryos reached the invagination stage; and some reached the enlargement stage with active photosynthetic tissues. However there were no to generation of complete plant regardless of additional treatment, such as "osmotic shock" or the additions of GA3--At present do not have an explanations for this results. Therefore, additional experiment should be in early, intermediate and later stages of somatic embryogenesis, in order to understand the mechanisms underlying the lack of development of plants from somatics embryos. PMID:11220222

  11. Molecular Aspects of Conifer Zygotic and Somatic Embryo Development: A Review of Genome-Wide Approaches and Recent Insights.

    PubMed

    Trontin, Jean-François; Klimaszewska, Krystyna; Morel, Alexandre; Hargreaves, Catherine; Lelu-Walter, Marie-Anne

    2016-01-01

    Genome-wide profiling (transcriptomics, proteomics, metabolomics) is providing unprecedented opportunities to unravel the complexity of coordinated gene expression during embryo development in trees, especially conifer species harboring "giga-genome." This knowledge should be critical for the efficient delivery of improved varieties through seeds and/or somatic embryos in fluctuating markets and to cope with climate change. We reviewed "omics" as well as targeted gene expression studies during both somatic and zygotic embryo development in conifers and tentatively puzzled over the critical processes and genes involved at the specific developmental and transition stages. Current limitations to the interpretation of these large datasets are going to be lifted through the ongoing development of comprehensive genome resources in conifers. Nevertheless omics already confirmed that master regulators (e.g., transcription and epigenetic factors) play central roles. As in model angiosperms, the molecular regulation from early to late embryogenesis may mainly arise from spatiotemporal modulation of auxin-, gibberellin-, and abscisic acid-mediated responses. Omics also showed the potential for the development of tools to assess the progress of embryo development or to build genotype-independent, predictive models of embryogenesis-specific characteristics. PMID:26619863

  12. [Somatic consequences of cannabis use].

    PubMed

    Cottencin, Olivier; Bence, Camille; Rolland, Benjamin; Karila, Laurent

    2013-12-01

    Cannabis can have negative effects in its users, and a range of acute and chronic health problems associated with cannabis use has been dentified. Acute cannabis consumption is rarely lethal but it is associated with an increased risk of motor vehicle accident because of longer reaction time or impaired motor coordination. Chronic effects of cannabis use include generally cardiovascular and respiratory consequences but there are also oral, gastrointestinal, cutaneous and mucous, metabolic, gynecologic and obstetrical, sexual consequences, and cancer But associated tobacco smoking or other potential confounders may explain part of those somatic consequences. PMID:24579346

  13. Immunological detection of potential signal-transduction proteins expressed during wheat somatic tissue culture.

    PubMed Central

    Nato, A; Mirshahi, A; Tichtinsky, G; Mirshahi, M; Faure, J P; Lavergne, D; De Buyser, J; Jean, C; Ducreux, G; Henry, Y

    1997-01-01

    An immunochemical approach was used to detect the expression of putative guanine nucleotide-binding proteins (G-proteins), arrestin, and nucleoside diphosphate kinases during wheat (Triticum aestivum) tissue culture initiated from immature embryos. Both the soluble and membrane extracts from the immature embryos revealed bands of 58, 40, and 16 kD with antibodies to G-protein (alpha subunit), arrestin, and nucleoside diphosphate kinase, respectively. These proteins were overexpressed in vitro in both nonembryogenic callus and embryogenic cultures. An additional soluble protein (32 kD) was detected by anti-G alpha antibodies in cultured tissues but not in immature embryos, suggesting a possible function in cell multiplication. Moreover, somatic embryogenesis was associated with the appearance of a 29-kD protein reactive with anti-arrstin antibodies, both in soluble and membrane fractions. Tissue-cultured genetic stocks of Chinese Spring wheat, including the disomic, 36 ditelosomic, and 6 nullisomic-tetrasomic wheat lines, were used to ascertain the chromosomal location of the genes encoding the 29-kD arrestin-like protein. The lack of a signal with the nonembryogenic ditelosomic 3 D short chromosome arm line suggests that the 3 D long chromosome arm possesses at least one gene involved in the expression of the 29-kD protein. The putative role of the 29-kD protein in signal-transduction regulating embryogenesis is discussed. PMID:9085574

  14. Epithelial self-organization in fruit fly embryogenesis

    NASA Astrophysics Data System (ADS)

    Hutson, M. Shane

    2010-03-01

    During fruit fly embryogenesis, there are several morphogenetic events in which sheets of epithelial cells expand, contract and bend due to coordinated intra- and intercellular forces. This tissue-level reshaping is accompanied by changes in the shape and arrangement of individual cells -- changes that can be measured quantitatively and dynamically using modern live-cell imaging techniques. Such data sets represent rich targets for computational modeling of self-organization; however, reproducing the observed cell- and tissue-level reshaping is not enough. The inverse problem of using cell shape changes to determine cell-level forces is ill-posed -- yielding non-unique solutions that cannot discriminate between active changes in cell shape and passive deformation. These non-unique solutions can be tested experimentally using in vivo laser-microsurgery -- i.e., cutting a targeted region of an epithelium and carefully tracking the temporal and spatial dependence of the subsequent strain relaxation. This technique uses a variety of incisions (hole, line or closed curve) to probe different aspects of epithelial mechanics: the local mesoscopic strain; the distribution of intracellular forces; changes in the cell-level power-law rheology; and the question of active versus passive deformation. I will discuss my group's work using laser-microsurgery to investigate two morphogenetic events in fruit fly embryogenesis: germband retraction and dorsal closure. In both cases, we find a substantial active mechanical role for the amnioserosa -- an epithelium that undergoes apoptosis near the end of embryogenesis and makes no part of the fly larva -- in reshaping an adjacent epithelium that becomes the larval epidermis. In these examples, self-organization of the fly embryo relies not only on self-organization of individual tissues, but also on the mechanical interactions between tissues.

  15. Systematic identification of long noncoding RNAs expressed during zebrafish embryogenesis

    PubMed Central

    Pauli, Andrea; Valen, Eivind; Lin, Michael F.; Garber, Manuel; Vastenhouw, Nadine L.; Levin, Joshua Z.; Fan, Lin; Sandelin, Albin; Rinn, John L.; Regev, Aviv; Schier, Alexander F.

    2012-01-01

    Long noncoding RNAs (lncRNAs) comprise a diverse class of transcripts that structurally resemble mRNAs but do not encode proteins. Recent genome-wide studies in humans and the mouse have annotated lncRNAs expressed in cell lines and adult tissues, but a systematic analysis of lncRNAs expressed during vertebrate embryogenesis has been elusive. To identify lncRNAs with potential functions in vertebrate embryogenesis, we performed a time-series of RNA-seq experiments at eight stages during early zebrafish development. We reconstructed 56,535 high-confidence transcripts in 28,912 loci, recovering the vast majority of expressed RefSeq transcripts while identifying thousands of novel isoforms and expressed loci. We defined a stringent set of 1133 noncoding multi-exonic transcripts expressed during embryogenesis. These include long intergenic ncRNAs (lincRNAs), intronic overlapping lncRNAs, exonic antisense overlapping lncRNAs, and precursors for small RNAs (sRNAs). Zebrafish lncRNAs share many of the characteristics of their mammalian counterparts: relatively short length, low exon number, low expression, and conservation levels comparable to that of introns. Subsets of lncRNAs carry chromatin signatures characteristic of genes with developmental functions. The temporal expression profile of lncRNAs revealed two novel properties: lncRNAs are expressed in narrower time windows than are protein-coding genes and are specifically enriched in early-stage embryos. In addition, several lncRNAs show tissue-specific expression and distinct subcellular localization patterns. Integrative computational analyses associated individual lncRNAs with specific pathways and functions, ranging from cell cycle regulation to morphogenesis. Our study provides the first systematic identification of lncRNAs in a vertebrate embryo and forms the foundation for future genetic, genomic, and evolutionary studies. PMID:22110045

  16. Stability of potato (Solanum tuberosum L.) plants regenerated via somatic embryos, axillary bud proliferated shoots, microtubers and true potato seeds: a comparative phenotypic, cytogenetic and molecular assessment.

    PubMed

    Sharma, Sanjeev Kumar; Bryan, Glenn J; Winfield, Mark O; Millam, Steve

    2007-11-01

    The stability, both genetic and phenotypic, of potato (Solanum tuberosum L.) cultivar Desiree plants derived from alternative propagation methodologies has been compared. Plants obtained through three clonal propagation routes-axillary-bud-proliferation, microtuberisation and a novel somatic embryogenesis system, and through true potato seeds (TPS) produced by selfing were evaluated at three levels: gross phenotype and minituber yield, changes in ploidy (measured by flow cytometry) and by molecular marker analysis [measured using AFLP (amplified fragment length polymorphism)]. The clonally propagated plants exhibited no phenotypic variation while the TPS-derived plants showed obvious phenotypic segregation. Significant differences were observed with respect to minituber yield while average plant height, at the time of harvesting, was not significantly different among plants propagated through four different routes. None of the plant types varied with respect to gross genome constitution as assessed by flow cytometry. However, a very low level of AFLP marker profile variation was seen amongst the somatic embryo (3 out of 451 bands) and microtuber (2 out of 451 bands) derived plants. Intriguingly, only AFLP markers generated using methylation sensitive restriction enzymes were found to show polymorphism. No polymorphism was observed in plants regenerated through axillary-bud-proliferation. The low level of molecular variation observed could be significant on a genome-wide scale, and is discussed in the context of possible methylation changes occurring during the process of somatic embryogenesis. PMID:17668235

  17. The use of centrifugation to study early Drosophila embryogenesis

    NASA Technical Reports Server (NTRS)

    Abbott, M. K.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    By the end of 10th nuclear cycle, the somatic nuclei of the Drosophila embryo have migrated to the periphery of the egg. Centrifugation of embryos did not result in the displacement of these nuclei, since cytoskeletal elements anchor them to the cortex. But, mild centrifugal forces displace the centrally located, nascent yolk nuclei. If this increased sensitivity to hypergravity occurs before the beginning of nuclear differentiation during cycle 8, when the nascent yolk and somatic nuclei physically separate, then it would mark the earliest functional difference between these two lineages.

  18. Hemoglobin regulation of plant embryogenesis and plant pathogen interaction

    PubMed Central

    Wally, Owen S.D.; Mira, Mohamed M.; Hill, Robert D.; Stasolla, Claudio

    2013-01-01

    Plant hemoglobins are ubiquitous molecules involved in several aspects of plant development and stress responses. Studies on the functional aspects of plant hemoglobins at the cellular level in these processes are limited, despite their ability to scavenge nitric oxide (NO), an important signal molecule interfering with hormone synthesis and sensitivity. This mini-review summarizes current knowledge on plant hemoglobins, analyzes their participation in plant pathogen interaction and embryogenesis and proposes a possible model centering on jasmonic acid (JA) as a downstream component of hemoglobin responses. PMID:23759548

  19. Perfluoroheptanoic acid affects amphibian embryogenesis by inducing the phosphorylation of ERK and JNK.

    PubMed

    Kim, Miran; Park, Mi Seon; Son, Jungeun; Park, Inji; Lee, Hyun-Kyung; Kim, Chowon; Min, Byung-Hwa; Ryoo, Jaewoong; Choi, Kwang Shik; Lee, Dong-Seok; Lee, Hyun-Shik

    2015-12-01

    Perfluoroalkyl compounds (PFCs) are globally distributed synthetic compounds that are known to adversely affect human health. Developmental toxicity assessment of PFCs is important to facilitate the evaluation of their environmental impact. In the present study, we assessed the developmental toxicity and teratogenicity of PFCs with different numbers of carbon atoms on Xenopus embryogenesis. An initial frog embryo teratogenicity assay-Xenopus (FETAX) assay was performed that identified perfluorohexanoic (PFHxA) and perfluoroheptanoic (PFHpA) acids as potential teratogens and developmental toxicants. The mechanism underlying this teratogenicity was also investigated by measuring the expression of tissue-specific biomarkers such as phosphotyrosine‑binding protein, xPTB (liver); NKX2.5 (heart); and Cyl18 (intestine). Whole‑mount in situ hybridization, reverse transcriptase‑polymerase chain reaction (RT-PCR), and histologic analyses detected severe defects in the liver and heart following exposure to PFHxA or PFHpA. In addition, immunoblotting revealed that PFHpA significantly increased the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), while PFHxA slightly increased these, as compared with the control. These results suggest that PFHxA and PFHpA are developmental toxicants and teratogens, with PFHpA producing more severe effects on liver and heart development through the induction of ERK and JNK phosphorylation. PMID:26459765

  20. The molecular, cellular, and morphological components of blood-brain barrier development during embryogenesis.

    PubMed

    Hagan, Nellwyn; Ben-Zvi, Ayal

    2015-02-01

    The blood brain barrier (BBB) is a hallmark of blood vessels in the brain and functions to protect the brain from unwanted blood born materials, support the unique metabolic needs of the brain, and define a stable environment crucial for brain homeostasis. The temporal profile of BBB development was long debated until recent studies produced convincing evidence demonstrating that the BBB is established and functional during embryogenesis. Here we review research focused on the molecular, cellular and morphological characteristics of BBB development. Our review discusses the precise temporal profile of BBB formation, the development of endothelial cell ultrastructure and the molecular components that provide sealing and transporting properties, the molecular pathways involved in the induction of BBB specific endothelial cell differentiation, the signaling pathways driving developmental angiogenesis versus barrier-genesis, and finally the contribution of other cell types to BBB formation. We examine aspects of BBB development that are still unresolved while highlighting research tools that could provide new insight to answer these open questions. PMID:25550218

  1. Transposable elements and the dynamic somatic genome

    PubMed Central

    Collier, Lara S; Largaespada, David A

    2007-01-01

    Although alterations in the genomes of somatic cells cannot be passed on to future generations, they can have beneficial or detrimental effects on the host organism, depending on the context in which they occur. This review outlines the ways in which transposable elements have important consequences for somatic cell genomes. PMID:18047697

  2. Depression, Life Events and Somatic Symptoms.

    ERIC Educational Resources Information Center

    Rozzini, Renzo; And Others

    1988-01-01

    Investigated the relationship between somatic symptoms, depression, and life events (health status, function, social satisfaction, income) in a population of 1,201 elderly persons living at home. Found depression was the most important factor in the appearance of somatic complaints; however, life events were important cofactors in defining…

  3. Arsenic exposure to killifish during embryogenesis alters muscle development.

    PubMed

    Gaworecki, Kristen M; Chapman, Robert W; Neely, Marion G; D'Amico, Angela R; Bain, Lisa J

    2012-02-01

    Epidemiological studies have correlated arsenic exposure in drinking water with adverse developmental outcomes such as stillbirths, spontaneous abortions, neonatal mortality, low birth weight, delays in the use of musculature, and altered locomotor activity. Killifish (Fundulus heteroclitus) were used as a model to help to determine the mechanisms by which arsenic could impact development. Killifish embryos were exposed to three different sodium arsenite concentrations and were collected at 32 h post-fertilization (hpf), 42 hpf, 168 hpf, or < 24 h post-hatch. A killifish oligo microarray was developed and used to examine gene expression changes between control and 25-ppm arsenic-exposed hatchlings. With artificial neural network analysis of the transcriptomic data, accurate prediction of each group (control vs. arsenic-exposed embryos) was obtained using a small subset of only 332 genes. The genes differentially expressed include those involved in cell cycle, development, ubiquitination, and the musculature. Several of the genes involved in cell cycle regulation and muscle formation, such as fetuin B, cyclin D-binding protein 1, and CapZ, were differentially expressed in the embryos in a time- and dose-dependent manner. Examining muscle structure in the hatchlings showed that arsenic exposure during embryogenesis significantly reduces the average muscle fiber size, which is coupled with a significant 2.1- and 1.6-fold upregulation of skeletal myosin light and heavy chains, respectively. These findings collectively indicate that arsenic exposure during embryogenesis can initiate molecular changes that appear to lead to aberrant muscle formation. PMID:22058191

  4. Specific nanotoxicity of graphene oxide during zebrafish embryogenesis.

    PubMed

    Chen, Yuming; Hu, Xiangang; Sun, Jing; Zhou, Qixing

    2016-02-01

    Graphene oxide (GO) has shown great potential for biological, medical, energy and electronic applications. As a consequence of these diverse applications, GO release into the ecosystem is inevitable; however, the corresponding risks are largely unknown, particularly with respect to the critical period of embryogenesis. This study revealed that GO adhered to and enveloped the chorion of zebrafish embryos mainly via hydroxyl group interactions, blocked the pore canals of the chorionic membrane, and caused marked hypoxia and hatching delay. Furthermore, GO spontaneously penetrated the chorion, entered the embryo via endocytosis, damaged the mitochondria and primarily translocated to the eye, heart and yolk sac regions, which are involved in the circulatory system of zebrafish. In these organs, GO induced excessive generation of reactive oxygen species and increased oxidative stress, DNA damage and apoptosis. Graphene oxide also induced developmental malformation of the eye, cardiac/yolk sac edema, tail flexure and heart rate reduction. In contrast to the common dose-effect relationships of nanoparticles, the adverse effects of GO on heart rate and tail/spinal cord flexure increased and then decreased as the GO concentration increased. These findings emphasize the specific adverse effects of GO on embryogenesis and highlight the potential ecological and health risks of GO. PMID:25704117

  5. Regulation of germ layer formation by pluripotency factors during embryogenesis

    PubMed Central

    2013-01-01

    The classical pluripotency factors Oct4, Klf4, Sox2, and Nanog are required for the maintenance of pluripotency and self-renewal of embryonic stem (ES) cells and can reprogram terminally differentiated cells into a pluripotent state. Alteration in the levels of these factors in ES cells will cause differentiation into different lineages, suggesting that they are critical determinants of cell fates. These factors show dynamic expression patterns during embryogenesis, in particular in the pluripotent or multipotent cells of an early stage embryo, implying that they are involved in the cell fate decision during early embryonic development. Functions and the underlying molecular mechanisms have been extensively studied for these factors in ES cells under cultured conditions. However, this does not mean that the results also hold true for intact embryos. In the review, I have summarized and discussed the findings on the functions and the underlying mechanisms of the classical pluripotency factors during early embryogenesis, in particular during germ layer formation. PMID:23497659

  6. Autophagy and apoptosis are redundantly required for C. elegans embryogenesis.

    PubMed

    Borsos, Eva; Erdélyi, Péter; Vellai, Tibor

    2011-05-01

    Apoptosis, the main form of regulated (or programmed) cell death, allows the organism to tightly control cell numbers and tissue size, and to protect itself from potentially damaging cells. This type of cellular self-killing has long been assumed to be essential for early development. In the nematode Caenorhabditis elegans, however, the core apoptotic cell death pathway appears to be dispensable for embryogenesis when most developmental cell deaths take place: mutant nematodes defective for apoptosis develop into adulthood, with superficially normal morphology and behavior. Accumulating evidence indicates a similar situation in mammalian systems as well. For example, apoptosis-deficient mice can grow as healthy, fertile adults. These observations raise the possibility that alternative cell death mechanisms may compensate for the lack of apoptotic machinery in developing embryos. Interestingly, C. elegans embryogenesis can also occur without autophagy, an alternative form of cellular self-destruction (also called autophagic cell death). In an upcoming paper we report that simultaneous inactivation of the autophagic and apoptotic gene cascades in C. elegans arrests development at early stages, and the affected embryos exhibit severe morphological defects. Double-mutant nematode embryos deficient in both autophagy and apoptosis are unable to undergo body elongation or to arrange several tissues correctly. This novel function of autophagy genes in morphogenesis indicates a more fundamental role for cellular self-digestion in tissue patterning than previously thought. PMID:21285529

  7. Somatic Cell Counts in Bovine Milk

    PubMed Central

    Dohoo, I. R.; Meek, A. H.

    1982-01-01

    Factors which influence somatic cell counts in bovine milk are reviewed and guidelines for their interpretation are presented. It is suggested that the thresholds of 300 000 and 250 000 cells/mL be used to identify infected quarters and cows respectively. However, it is stressed that somatic cell counts are general indicators of udder health which are subject to the influence of many factors. Therefore the evaluation of several successive counts is preferable to the interpretation of an individual count. Relationships between somatic cell counts and both milk production and milk composition are discussed. Subclinical mastitis reduces milk quality and decreases yield although the relationship between production loss and somatic cell count requires clarification. Finally the availability of somatic cell counting programs in Canada is presented. PMID:17422127

  8. Somatic Mosaicism in the Human Genome

    PubMed Central

    Freed, Donald; Stevens, Eric L.; Pevsner, Jonathan

    2014-01-01

    Somatic mosaicism refers to the occurrence of two genetically distinct populations of cells within an individual, derived from a postzygotic mutation. In contrast to inherited mutations, somatic mosaic mutations may affect only a portion of the body and are not transmitted to progeny. These mutations affect varying genomic sizes ranging from single nucleotides to entire chromosomes and have been implicated in disease, most prominently cancer. The phenotypic consequences of somatic mosaicism are dependent upon many factors including the developmental time at which the mutation occurs, the areas of the body that are affected, and the pathophysiological effect(s) of the mutation. The advent of second-generation sequencing technologies has augmented existing array-based and cytogenetic approaches for the identification of somatic mutations. We outline the strengths and weaknesses of these techniques and highlight recent insights into the role of somatic mosaicism in causing cancer, neurodegenerative, monogenic, and complex disease. PMID:25513881

  9. Bone Morphogenetic Protein 15 (BMP15) Acts as a BMP and Wnt Inhibitor during Early Embryogenesis*

    PubMed Central

    Di Pasquale, Elisa; Brivanlou, Ali H.

    2009-01-01

    Bone morphogenetic protein 15 (BMP15) belongs to an unusual subgroup of the transforming growth factor β (TGFβ) superfamily of signaling ligands as it lacks a key cysteine residue in the mature region required for proper intermolecular dimerization. Naturally occurring BMP15 mutation leads to early ovarian failure in humans, and BMP15 has been shown to activate the Smad1/5/8 pathway in that context. Despite its important role in germ cell specification, the embryological function of BMP15 remains unknown. Surprisingly, we find that during early Xenopus embryogenesis BMP15 acts solely as an inhibitor of the Smad1/5/8 pathway and the Wnt pathway. BMP15 gain-of-function leads to embryos with secondary ectopic heads and to direct neural induction in intact explants. BMP15 inhibits BMP4-mediated epidermal induction in dissociated explants. BMP15 strongly inhibits BRE response induced by BMP4 and blocks phosphorylation and activation of Smad1/5/8 MH2-domain. Mechanistically, BMP15 protein specifically interacts with BMP4 protein, suggesting inhibition upstream of receptor binding. Loss-of-function experiments using morpholinos or a naturally occurring human BMP15 dominant-negative mutant (BMP15-Y235C) leads to embryos lacking head. BMP15-Y235C also eliminates the inhibitory activity of BMP15 on BRE (BMP-responsive element). Finally, we show that BMP15 inhibits the canonical branch of the Wnt pathway, upstream of β-catenin. We, thus, demonstrate that BMP15 is necessary and sufficient for the specification of dorso-anterior structures and highlight novel mechanisms of BMP15 function that strongly suggest a reinterpretation of its function in ovaries specially for ovarian failure. PMID:19553676

  10. Comparative expression pattern analysis of WUSCHEL-related homeobox 2 (WOX2) and WOX8/9 in developing seeds and somatic embryos of the gymnosperm Picea abies.

    PubMed

    Palovaara, Joakim; Hallberg, Henrik; Stasolla, Claudio; Hakman, Inger

    2010-10-01

    • In seed plants, current knowledge concerning embryonic pattern formation by polar auxin transport (PAT) and WUSCHEL-related homeobox (WOX) gene activity is primarily derived from studies on angiosperms, while less is known about these processes in gymnosperms. In view of the differences in their embryogeny, and the fact that somatic embryogenesis is used for mass propagation of conifers, a better understanding of embryo development is vital. • The expression patterns of PaWOX2 and PaWOX8/9 were followed with quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH) during seed and somatic embryo development in Norway spruce (Picea abies), and in somatic embryos treated with the PAT inhibitor N-1-naphthylphthalamic acid (NPA). • Both PaWOX2 and PaWOX8/9 were highly expressed at the early growth stages of zygotic and somatic embryos, and shared a similar expression pattern over the entire embryo. At later embryo stages, high expression of PaWOX8/9 became restricted to cotyledon primordia, epidermis, procambium and root apical meristem (RAM), which became most evident in NPA-treated somatic embryos, while expression of PaWOX2 was much lower. • Our results suggest an ancestral role of WOX in seed plant embryo development, and strengthen the proposed connection between PAT, PIN-FORMED (PIN) and WOX in the regulation of embryo patterning in seed plants. PMID:20561212

  11. Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water availability: transcriptomic and proteomic analyses.

    PubMed

    Morel, Alexandre; Teyssier, Caroline; Trontin, Jean-François; Eliášová, Kateřina; Pešek, Bedřich; Beaufour, Martine; Morabito, Domenico; Boizot, Nathalie; Le Metté, Claire; Belal-Bessai, Leila; Reymond, Isabelle; Harvengt, Luc; Cadene, Martine; Corbineau, Françoise; Vágner, Martin; Label, Philippe; Lelu-Walter, Marie-Anne

    2014-09-01

    Maritime pine somatic embryos (SEs) require a reduction in water availability (high gellan gum concentration in the maturation medium) to reach the cotyledonary stage. This key switch, reported specifically for pine species, is not yet well understood. To facilitate the use of somatic embryogenesis for mass propagation of conifers, we need a better understanding of embryo development. Comparison of both transcriptome (Illumina RNA sequencing) and proteome [two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with mass spectrometry (MS) identification] of immature SEs, cultured on either high (9G) or low (4G) gellan gum concentration, was performed, together with analysis of water content, fresh and dry mass, endogenous abscisic acid (ABA; gas chromatography-MS), soluble sugars (high-pressure liquid chromatography), starch and confocal laser microscope observations. This multiscale, integrated analysis was used to unravel early molecular and physiological events involved in SE development. Under unfavorable conditions (4G), the glycolytic pathway was enhanced, possibly in relation to cell proliferation that may be antagonistic to SE development. Under favorable conditions (9G), SEs adapted to culture constraint by activating specific protective pathways, and ABA-mediated molecular and physiological responses promoting embryo development. Our results suggest that on 9G, germin-like protein and ubiquitin-protein ligase could be used as predictive markers of SE development, whereas protein phosphatase 2C could be a biomarker for culture adaptive responses. This is the first characterization of early molecular mechanisms involved in the development of pine SEs following an increase in gellan gum concentration in the maturation medium, and it is also the first report on somatic embryogenesis in conifers combining transcriptomic and proteomic datasets. PMID:24460664

  12. Somatic Treatments for Mood Disorders

    PubMed Central

    Rosa, Moacyr A; Lisanby, Sarah H

    2012-01-01

    Somatic treatments for mood disorders represent a class of interventions available either as a stand-alone option, or in combination with psychopharmacology and/or psychotherapy. Here, we review the currently available techniques, including those already in clinical use and those still under research. Techniques are grouped into the following categories: (1) seizure therapies, including electroconvulsive therapy and magnetic seizure therapy, (2) noninvasive techniques, including repetitive transcranial magnetic stimulation, transcranial direct current stimulation, and cranial electric stimulation, (3) surgical approaches, including vagus nerve stimulation, epidural electrical stimulation, and deep brain stimulation, and (4) technologies on the horizon. Additionally, we discuss novel approaches to the optimization of each treatment, and new techniques that are under active investigation. PMID:21976043

  13. Induction of a Reductive Pathway for Deoxyribonucleotide Synthesis during Early Embryogenesis of the Sea Urchin

    PubMed Central

    Noronha, John M.; Sheys, Gerald H.; Buchanan, John M.

    1972-01-01

    Cell-free extracts of Arbacia eggs (Arbacia punctulata) apparently do not contain an enzymatic system for the reduction of ribonucleotides to deoxyribonucleotides. However, during an interval of 5 hr after fertilization at 23°, an enzymatic system is produced that is capable of catalyzing the reduction of CDP to dCDP in the presence of Mg2+, ethylenediaminetetraacetate, ATP, and a reducing agent, dithiothreitol. The activity is first seen about 1 hr after fertilization, and reaches a peak at about 5 hr. The appearance of the ribonucleotide reductase is prevented by the addition of emetine or puromycin, inhibitors of protein synthesis, to the cells before fertilization. Inclusion of actinomycin D in the cell suspension at a concentration sufficient to inhibit synthesis of messenger RNA does not appreciably affect the production of the enzyme activity. Preexisting, maternal RNA is thus used for synthesis of reductase. Ribonucleotide reductase may, therefore, represent the first example of an enzyme system absent in unfertilized eggs that is produced in response to fertilization. PMID:4626400

  14. De novo DNA methylation through the 5'-segment of the H19 ICR maintains its imprint during early embryogenesis.

    PubMed

    Matsuzaki, Hitomi; Okamura, Eiichi; Takahashi, Takuya; Ushiki, Aki; Nakamura, Toshinobu; Nakano, Toru; Hata, Kenichiro; Fukamizu, Akiyoshi; Tanimoto, Keiji

    2015-11-15

    Genomic imprinting is a major monoallelic gene expression regulatory mechanism in mammals, and depends on gamete-specific DNA methylation of specialized cis-regulatory elements called imprinting control regions (ICRs). Allele-specific DNA methylation of the ICRs is faithfully maintained at the imprinted loci throughout development, even in early embryos where genomes undergo extensive epigenetic reprogramming, including DNA demethylation, to acquire totipotency. We previously found that an ectopically introduced H19 ICR fragment in transgenic mice acquired paternal allele-specific methylation in the somatic cells of offspring, whereas it was not methylated in sperm, suggesting that its gametic and postfertilization modifications were separable events. We hypothesized that this latter activity might contribute to maintenance of the methylation imprint in early embryos. Here, we demonstrate that methylation of the paternally inherited transgenic H19 ICR commences soon after fertilization in a maternal DNMT3A- and DNMT3L-dependent manner. When its germline methylation was partially obstructed by insertion of insulator sequences, the endogenous paternal H19 ICR also exhibited postfertilization methylation. Finally, we refined the responsible sequences for this activity in transgenic mice and found that deletion of the 5' segment of the endogenous paternal H19 ICR decreased its methylation after fertilization and attenuated Igf2 gene expression. These results demonstrate that this segment of the H19 ICR is essential for its de novo postfertilization DNA methylation, and that this activity contributes to the maintenance of imprinted methylation at the endogenous H19 ICR during early embryogenesis. PMID:26417043

  15. Somatic mosaicism of a novel IKBKG mutation in a male patient with incontinentia pigmenti.

    PubMed

    Hull, Sarah; Arno, Gavin; Thomson, Penelope; Mutch, Stacey; Webster, Andrew R; Rai, Harjeet; Hill, Virginia; Moore, Anthony T

    2015-07-01

    Incontinentia pigmenti (IP) is an X-linked, dominant genodermatosis usually fatal in utero in males. In rare circumstances, survival is possible due to abnormal karyotype or somatic mosaicism. In this report, the mechanism and significance of loss of detectable mutation in peripheral blood leukocytes of a somatic mosaic male is discussed and an alternative approach to achieving molecular diagnosis presented. A male patient is reported, who initially presented at 2 days of age with a rash and seizure. Clinical assessment and histology of a skin biopsy were consistent with a diagnosis of IP. He was subsequently found to have bilateral retinal detachments. Screening for the common deletion in IKBKG was negative. A novel nonsense variant, c.937C>T (p.Gln313*) in IKBKG was identified at an approximate level of 15% in a blood sample taken at 10 days of age, but was undetectable in a sample taken at 3 years most likely due to selective apoptosis of mutant cells. Samples taken from the patient when he was 5-6 years of age identified the mutation at a low level in hair root and urine but not in blood or buccal cells. The detection of the mutation in cells derived from all germ layers indicates a de novo event at an early stage of embryogenesis. This is the first report of a nonsense mutation in a male IP patient. PMID:25944529

  16. [Somatization disorder - an overdiagnosed but underestimated illness].

    PubMed

    Karvonen, Juha T; Läksy, Kristian; Räsänen, Sami

    2016-01-01

    Physical symptoms often occur in the absence of physical illness. This is termed somatization when the symptoms are caused by psychic factors. When abundant symptoms affect the functional capacity and cause subjective harm and seeking healthcare services, a psychic disorder may be in question. Somatization may be associated with numerous psychic disorders. It may, however, also be a question of a somatoform disorder having a physical symptom picture. Somatization disorder is one of the somatoform disorders. Recognition of the disorder is often the problem in its treatment. Establishing a long-term treatment relationship actually forms the basis for therapy. PMID:26951025

  17. TOPLESS mediates auxin-dependent transcriptional repression during Arabidopsis embryogenesis.

    PubMed

    Szemenyei, Heidi; Hannon, Mike; Long, Jeff A

    2008-03-01

    The transcriptional response to auxin is critical for root and vascular development during Arabidopsis embryogenesis. Auxin induces the degradation of AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) transcriptional repressors, freeing their binding partners, the AUXIN RESPONSE FACTOR (ARF) proteins, which can activate transcription of auxin response genes. We show that TOPLESS (TPL) can physically interact with IAA12/BODENLOS (IAA12/BDL) through an ETHYLENE RESPONSE FACTOR (ERF)-associated amphiphilic repression (EAR) motif. TPL can repress transcription in vivo and is required for IAA12/BDL repressive activity. In addition, tpl-1 can suppress the patterning defects of the bdl-1 mutant. Direct interaction between TPL and ARF5/MONOPTEROS, which is regulated by IAA12/BDL, results in a loss-of-function arf5/mp phenotype. These observations show that TPL is a transcriptional co-repressor and further our understanding of how auxin regulates transcription during plant development. PMID:18258861

  18. Embryogenesis and Plant Regeneration from Isolated Wheat Zygotes.

    PubMed

    Kumlehn, Jochen

    2016-01-01

    Wheat zygotes can be mechanically isolated and cultivated to continue their development in vitro. Since each zygote needs to be individually isolated, only relatively few of these cells are available per experiment. To facilitate embryonic growth despite of this limitation, the zygotes are kept within a culture insert placed in a larger dish which itself contains embryogenic pollen cocultivated for continuous medium conditioning. This setup ensures that the two cultures, while being physically separated from one another, can exchange essential intercellular signal molecules passing through the bottom of the insert which is made of a permeable membrane. Thanks to the natural fate of zygotes, which is to form an embryo followed by the generation of a plant, embryogenesis and plant regeneration are achieved at much higher efficiency as compared to other single-cell systems. While the method is largely independent of the genotype, it allows for the nondestructive observation, manipulation, and individual analysis of zygotes and very young embryos. PMID:26619884

  19. Developmental regulation of Tbx5 in zebrafish embryogenesis.

    PubMed

    Begemann, G; Ingham, P W

    2000-02-01

    T-box (tbx) genes constitute a large family of transcriptional regulators involved in developmental patterning processes. In tetrapods, tbx5 has been implicated in specifying forelimb type identity. Here, we report the cloning of the zebrafish tbx5.1 gene and characterise its expression during zebrafish embryogenesis and early larval development of wild type and mutant embryos that affect pectoral fin patterning. tbx5.1 is expressed during development of the heart, the pectoral fins and the eye. Notably, its expression in the lateral plate mesoderm defines a single and continuous region of heart and pectoral fin precursor cells, and constitutes the earliest specific marker for pectoral fin development in the zebrafish. PMID:10640716

  20. Membrane dynamics in mammalian embryogenesis: Implication in signal regulation.

    PubMed

    Wada, Yoh; Sun-Wada, Ge-Hong; Kawamura, Nobuyuki; Yasukawa, Jyunichiro

    2016-03-01

    Eukaryotes have evolved an array of membrane compartments constituting secretory and endocytic pathways that allow the flow of materials. Both pathways perform important regulatory roles. The secretory pathway is essential for the production of extracellular, secreted signal molecules, but its function is not restricted to a mere route connecting intra- and extracellular compartments. Post-translational modifications also play an integral function in the secretory pathway and are implicated in developmental regulation. The endocytic pathway serves as a platform for relaying signals from the extracellular stimuli to intracellular mediators, and then ultimately inducing signal termination. Here, we discuss recent studies showing that dysfunction in membrane dynamics causes patterning defects in embryogenesis and tissue morphogenesis in mammals. Birth Defects Research (Part C) 108:33-44, 2016. © 2016 Wiley Periodicals, Inc. PMID:26992153

  1. Differential expression of two scribble isoforms during Drosophila embryogenesis.

    PubMed

    Li, M; Marhold, J; Gatos, A; Török, I; Mechler, B M

    2001-10-01

    The tumour suppressor gene scribble (scrib) is required for epithelial polarity and growth control in Drosophila. Here, we report the identification and embryonic expression pattern of two Scrib protein isoforms resulting from alternative splicing during scrib transcription. Both proteins are first ubiquitously expressed during early embryogenesis. Then, during morphogenesis each Scrib protein displays a specific pattern of expression in the central and peripheral nervous systems, CNS and PNS, respectively. During germ band extension, the expression of the longer form Scrib1 occurs predominantly in the neuroblasts derived from the neuro-ectoderm and becomes later restricted to CNS neurones as well as to the pole cells in the gonads. By contrast, the shorter form Scrib2 is strongly expressed in the PNS and a subset of CNS neurones. PMID:11578873

  2. [Development of neuronal activity of telencephalic structures during chicken embryogenesis].

    PubMed

    Gevorgian, E G; Bogdanov, O V; Mikhaĭlenok, E L

    1980-01-01

    Microelectrophysiological studies have been made on the development of neuronal activity in the structures of the striatal complex 17--21-day chick embryos. The density of spontaneously active neurones, their frequency parameters, the pattern of impulse activity and presentation of synaptic inputs for different modalities were investigated. The data obtained indicate the existence of significant heterochrony in the onset of neuronal activity in different parts of the telencephalon: the development of neuronal systems in phylogenetically more ancient structures, i.e. archi- and paliostriatum--takes place earlier than in younger formations--neo- and hyperstriatum. It is concluded that at final stages of embryogenesis the structure of neuronal activity of motor analyser undergoes deep functional changes which provide for the necessary level of plasticity of functional brain systems accounting for adaptive changes in the motor activity during embryonic development. PMID:7405442

  3. Endothelial Erg expression is required for embryogenesis and vascular integrity

    PubMed Central

    Han, Rong; Pacifici, Maurizio; Iwamoto, Masahiro; Trojanowska, Maria

    2015-01-01

    abstract Members of the ETS family of transcription factors are involved in several developmental processes including endothelial cell specification and blood vessel formation, but their exact roles remain unclear. The family member Erg is highly expressed in endothelial cells as compared to other developing cell types including chondrocytes, hematopoietic cells and mesodermal cells. To study the specific roles ERG plays in endothelial cell specification and function during early embryogenesis, we conditionally ablated it by mating ErgloxP/loxP and Tie2-Cre mice. We found that mutant embryos died by mid-gestation and that angiogenesis and vascular integrity were highly compromised. Our study reveals that ERG has essential and cell autonomous roles in endothelial cell development and blood vessel maintenance. PMID:26061019

  4. Patterns and functions of STAT activation during Drosophila embryogenesis

    PubMed Central

    Li, Jinghong; Li, Wenjun; Calhoun, Healani C.; Xia, Fan; Gao, Fen-Biao; Li, Willis X.

    2011-01-01

    The JAK/STAT pathway mediates cytokine signaling in mammals and is involved in the function and development of the hematopoietic and immune systems. To investigate the biological functions of the JAK/STAT pathway during Drosophila development, we examined the tissue-specific localization of the tyrosine-phosphorylated, or activated form of Drosophila STAT, STAT92E. Here we show that during Drosophila embryonic development STAT92E activation is prominently detected in multiple tissues and in different developmental stages. These tissues include the tracheal pits, elongating intestinal tracks, and growing axons. We demonstrate that stat92E mutants are defective in tracheal formation, hindgut elongation, and nervous system development. Conversely, STAT92E overactivation caused premature development of the tracheal and nervous systems, and over-elongation of the hindgut. These results suggest that STAT activation is involved in proper differentiation and morphogenesis of multiple tissues during Drosophila embryogenesis. PMID:14654218

  5. Current insights into hormonal regulation of microspore embryogenesis

    PubMed Central

    Żur, Iwona; Dubas, Ewa; Krzewska, Monika; Janowiak, Franciszek

    2015-01-01

    Plant growth regulator (PGR) crosstalk and interaction with the plant’s genotype and environmental factors play a crucial role in microspore embryogenesis (ME), controlling microspore-derived embryo differentiation and development as well as haploid/doubled haploid plant regeneration. The complexity of the PGR network which could exist at the level of biosynthesis, distribution, gene expression or signaling pathways, renders the creation of an integrated model of ME-control crosstalk impossible at present. However, the analysis of the published data together with the results received recently with the use of modern analytical techniques brings new insights into hormonal regulation of this process. This review presents a short historical overview of the most important milestones in the recognition of hormonal requirements for effective ME in the most important crop plant species and complements it with new concepts that evolved over the last decade of ME studies. PMID:26113852

  6. Multiple roles for protein kinase C in gastropod embryogenesis.

    PubMed

    Zukaite, Vainora; Cook, Richard T; Walker, Anthony J

    2016-04-01

    Protein kinase C (PKC) contributes to the correct development of organisms, but its importance to the embryogenesis of molluscs is not yet known. We report here that PKC activation is cyclic within early developing embryos of the gastropod snail Lymnaea stagnalis, and that activation with phorbol myristate acetate (PMA) results in disorganised and developmentally arrested embryos within 24 h. Moreover, chronic modulation of PKC activation by PMA or by the PKC inhibitor GF109203X in early embryos results in altered rotation and gliding behaviours and heartbeat during development. Finally, dis-regulation of PKC activity during early development significantly increased the duration to hatching. Our findings thus support novel roles for PKC in L. stagnalis embryos, in several physiological contexts, providing further insights into the importance of protein kinases for gastropod development in general. PMID:26384249

  7. Genome-wide analysis of spatiotemporal gene expression patterns during early embryogenesis in rice.

    PubMed

    Itoh, Jun-Ichi; Sato, Yutaka; Sato, Yutaka; Hibara, Ken-Ichiro; Shimizu-Sato, Sae; Kobayashi, Hiromi; Takehisa, Hinako; Sanguinet, Karen A; Namiki, Nobukazu; Nagamura, Yoshiaki

    2016-04-01

    Embryogenesis in rice is different from that of most dicotolydonous plants in that it shows a non-stereotypic cell division pattern, formation of dorsal-ventral polarity, and endogenous initiation of the radicle. To reveal the transcriptional features associated with developmental events during rice early embryogenesis, we used microarray analysis coupled with laser microdissection to obtain both spatial and temporal transcription profiles. Our results allowed us to determine spatial expression foci for each expressed gene in the globular embryo, which revealed the importance of phytohormone-related genes and a suite of transcription factors to early embryogenesis. Our analysis showed the polarized expression of a small number of genes along the apical-basal and dorsal-ventral axes in the globular embryo, which tended to fluctuate in later developmental stages. We also analyzed gene expression patterns in the early globular embryo and how this relates to expression in embryonic organs at later stages. We confirmed the accuracy of the expression patterns found by microarray analysis of embryo subdomains usingin situhybridization. Our study identified homologous genes fromArabidopsis thalianawith known functions in embryogenesis in addition to unique and uncharacterized genes that show polarized expression patterns during embryogenesis. The results of this study are presented in a database to provide a framework for spatiotemporal gene expression during rice embryogenesis, to serve as a resource for future functional analysis of genes, and as a basis for comparative studies of plant embryogenesis. PMID:26903508

  8. Eyelid Closure in Embryogenesis Is Required for Ocular Adnexa Development

    PubMed Central

    Meng, Qinghang; Mongan, Maureen; Carreira, Vinicius; Kurita, Hisaka; Liu, Chia-yang; Kao, Winston W.-Y.; Xia, Ying

    2014-01-01

    Purpose. Mammalian eye development requires temporary fusion of the upper and lower eyelids in embryogenesis. Failure of lid closure in mice leads to an eye open at birth (EOB) phenotype. Many genetic mutant strains develop this phenotype and studies of the mutants lead to a better understanding of the signaling mechanisms of morphogenesis. The present study investigates the roles of lid closure in eye development. Methods. Seven mutant mouse strains were generated by different gene ablation strategies that inactivated distinct signaling pathways. These mice, including systemic ablation of Map3k1 and Dkk2, ocular surface epithelium (OSE) knockout of c-Jun and Egfr, conditional knockout of Shp2 in stratified epithelium (SE), as well as the Map3k1/Jnk1 and Map3k1/Rhoa compound mutants, all exhibited defective eyelid closure. The embryonic and postnatal eyes in these mice were characterized by histology and immunohistochemistry. Results. Some eye abnormalities, such as smaller lens in the Map3k1-null mice and Harderian gland hypoplasia in the Dkk2-null mice, appeared to be mutant strain–specific, whereas other abnormalities were seen in all mutants examined. The common defects included corneal erosion/ulceration, meibomian gland hypoplasia, truncation of the eyelid tarsal muscles, failure of levator palpebrae superioris (LPS) extension into the upper eyelid and misplacement of the inferior oblique (IO) muscle and inferior rectus (IR) muscle. The muscle defects were traced to the prenatal fetuses. Conclusions. In addition to providing a protective barrier for the ocular surface, eyelid closure in embryogenesis is required for the development of ocular adnexa, including eyelid and extraocular muscles. PMID:25377219

  9. The expression and roles of parent-of-origin genes in early embryogenesis of angiosperms

    PubMed Central

    Luo, An; Shi, Ce; Zhang, Liyao; Sun, Meng-Xiang

    2014-01-01

    Uniparental transcripts during embryogenesis may arise due to gamete delivery during fertilization or genomic imprinting. Such transcripts have been found in a number of plant species and appear critical for the early development of embryo or endosperm in seeds. Although the regulatory expression mechanism and function of these genes in embryogenesis require further elucidation, recent studies suggest stage-specific and highly dynamic features that might be essential for critical developmental events such as zygotic division and cell fate determination during embryogenesis. Here, we summarize the current work in this field and discuss future research directions. PMID:25566300

  10. Somatic Complaints in Anxious Youth

    PubMed Central

    Crawley, Sarah A.; Caporino, Nicole E.; Birmaher, Boris; Ginsburg, Golda; Piacentini, John; Albano, Anne Marie; Sherrill, Joel; Sakolsky, Dara; Compton, Scott N.; Rynn, Moira; McCracken, James; Gosch, Elizabeth; Keeton, Courtney; March, John; Walkup, John T.; Kendall, Philip C.

    2013-01-01

    This study examined (a) demographic and clinical characteristics associated with physical symptoms in anxiety-disordered youth and (b) the impact of cognitive-behavioral therapy (Coping Cat), medication (sertraline), their combination, and pill placebo on physical symptoms. Youth (N = 488, ages 7–17 years) with a principal diagnosis of generalized anxiety disorder, separation anxiety disorder, or social phobia participated as part of a multi-site, randomized controlled trial and received treatment delivered over 12 weeks. Diagnostic status, symptom severity, and impairment were assessed at baseline and week 12. The total number and severity of physical symptoms was associated with age, principal diagnosis, anxiety severity, impairment, and the presence of comorbid internalizing disorders. Common somatic complaints were headaches, stomachaches, head cold or sniffles, sleeplessness, and feeling drowsy or too sleepy. Physical symptoms decreased over the course of treatment, and were unrelated to treatment condition. Clinical implications and directions for future research are discussed. (ClinicalTrials.gov number, NCT00052078) PMID:24129543

  11. Pilates, Mindfulness and Somatic Education

    PubMed Central

    Caldwell, Karen; Quin, Rebecca; Harrison, Mandy; Greeson, Jeffrey

    2014-01-01

    The Pilates Method is a form of somatic education with the potential to cultivate mindfulness – a mental quality associated with overall well-being. However, controlled studies are needed to determine whether changes in mindfulness are specific to the Pilates Method or also result from other forms of exercise. This quasi-experimental study compared Pilates Method mat classes and recreational exercise classes on measures of mindfulness and well-being at the beginning, middle and end of a 15 week semester. Total mindfulness scores increased overall for the Pilates Method group but not for the exercise control group, and these increases were directly related to end of semester ratings of self-regulatory self-efficacy, perceived stress and mood. Findings suggest that the Pilates Method specifically enhances mindfulness, and these increases are associated with other measures of wellness. The changes in mindfulness identified in this study support the role of the Pilates Method in the mental well-being of its practitioners and its potential to support dancers’ overall well-being. PMID:25328542

  12. Somatic complaints in anxious youth.

    PubMed

    Crawley, Sarah A; Caporino, Nicole E; Birmaher, Boris; Ginsburg, Golda; Piacentini, John; Albano, Anne Marie; Sherrill, Joel; Sakolsky, Dara; Compton, Scott N; Rynn, Moira; McCracken, James; Gosch, Elizabeth; Keeton, Courtney; March, John; Walkup, John T; Kendall, Philip C

    2014-08-01

    This study examined (a) demographic and clinical characteristics associated with physical symptoms in anxiety-disordered youth and (b) the impact of cognitive-behavioral therapy (Coping Cat), medication (sertraline), their combination, and pill placebo on physical symptoms. Youth (N = 488, ages 7-17 years) with a principal diagnosis of generalized anxiety disorder, separation anxiety disorder, or social phobia participated as part of a multi-site, randomized controlled trial and received treatment delivered over 12 weeks. Diagnostic status, symptom severity, and impairment were assessed at baseline and week 12. The total number and severity of physical symptoms was associated with age, principal diagnosis, anxiety severity, impairment, and the presence of comorbid internalizing disorders. Common somatic complaints were headaches, stomachaches, head cold or sniffles, sleeplessness, and feeling drowsy or too sleepy. Physical symptoms decreased over the course of treatment, and were unrelated to treatment condition. Clinical implications and directions for future research are discussed (ClinicalTrials.gov number, NCT00052078). PMID:24129543

  13. Processed pseudogenes acquired somatically during cancer development.

    PubMed

    Cooke, Susanna L; Shlien, Adam; Marshall, John; Pipinikas, Christodoulos P; Martincorena, Inigo; Tubio, Jose M C; Li, Yilong; Menzies, Andrew; Mudie, Laura; Ramakrishna, Manasa; Yates, Lucy; Davies, Helen; Bolli, Niccolo; Bignell, Graham R; Tarpey, Patrick S; Behjati, Sam; Nik-Zainal, Serena; Papaemmanuil, Elli; Teixeira, Vitor H; Raine, Keiran; O'Meara, Sarah; Dodoran, Maryam S; Teague, Jon W; Butler, Adam P; Iacobuzio-Donahue, Christine; Santarius, Thomas; Grundy, Richard G; Malkin, David; Greaves, Mel; Munshi, Nikhil; Flanagan, Adrienne M; Bowtell, David; Martin, Sancha; Larsimont, Denis; Reis-Filho, Jorge S; Boussioutas, Alex; Taylor, Jack A; Hayes, Neil D; Janes, Sam M; Futreal, P Andrew; Stratton, Michael R; McDermott, Ultan; Campbell, Peter J

    2014-01-01

    Cancer evolves by mutation, with somatic reactivation of retrotransposons being one such mutational process. Germline retrotransposition can cause processed pseudogenes, but whether this occurs somatically has not been evaluated. Here we screen sequencing data from 660 cancer samples for somatically acquired pseudogenes. We find 42 events in 17 samples, especially non-small cell lung cancer (5/27) and colorectal cancer (2/11). Genomic features mirror those of germline LINE element retrotranspositions, with frequent target-site duplications (67%), consensus TTTTAA sites at insertion points, inverted rearrangements (21%), 5' truncation (74%) and polyA tails (88%). Transcriptional consequences include expression of pseudogenes from UTRs or introns of target genes. In addition, a somatic pseudogene that integrated into the promoter and first exon of the tumour suppressor gene, MGA, abrogated expression from that allele. Thus, formation of processed pseudogenes represents a new class of mutation occurring during cancer development, with potentially diverse functional consequences depending on genomic context. PMID:24714652

  14. Somatization: a borderland between medicine and psychiatry.

    PubMed Central

    Lipowski, Z J

    1986-01-01

    Somatization is the tendency to experience and communicate psychologic distress in the form of somatic symptoms that the patient misinterprets as signifying serious physical illness. Patients with persistent somatization relentlessly seek medical diagnosis and treatment despite repeated reassurance that physical illness is either absent or insufficient to account for their symptoms and disability. Such abnormal illness behaviour leads to overuse of health care facilities and contributes to the high cost of health care. Somatization may occur transiently in response to stressful life events or it may be persistent and result in chronic partial or total disability. Diagnostic and therapeutic guidelines that may help physicians identify and manage such patients more effectively are discussed. PMID:3489512

  15. Stress and Somatic Complaints in Low-Income Urban Adolescents.

    ERIC Educational Resources Information Center

    Reynolds, Linda K.; O'Koon, Jeffrey H.; Papademetriou, Eros; Szczygiel, Sylvia; Grant, Kathryn E.

    2001-01-01

    Studied rates of somatic complaints and the association between stress and somatic complaints for 1,030 low-income urban adolescents in grades 6 through 8. For both boys and girls, somatization was the most commonly reported internalizing symptom, and heightened rates of urban stress predicted heightened rates of somatic complaints. (SLD)

  16. Predictors for somatic symptoms in children.

    PubMed

    Banks, Kerry; Bevan, Ann

    2014-02-01

    This literature review focuses on risk factors that influence somatic symptoms in children and young people aged between one and 18 years. Relevant risk factors identified were: past and current emotions, personality types and coping styles, poor environment including trauma and stress, and parental influences. It was found that most somatic complaints are caused by a combination of these and other risk factors, demonstrating the complexity of this diagnosis. PMID:24517619

  17. (Somatic mutations in nuclear and mitochondrial DNA)

    SciTech Connect

    Not Available

    1992-01-01

    The study is concerned the design of new assays that may detect rare somatic mutations in nuclear and mitochondrial DNA, which may increase upon exposure to mutagens, and thus become a marker of human exposure to such mutagens. Two assays for somatic mutation were presented, one for mitochondrial DNA deletions which was developed by the author, and one for deletions of the ADA gene which resides in the nucleus.

  18. Somatization disorder in a university hospital.

    PubMed

    deGruy, F; Crider, J; Hashimi, D K; Dickinson, P; Mullins, H C; Troncale, J

    1987-12-01

    Somatization disorder is a condition characterized by multiple unexplained complaints. To characterize this disorder as it occurs in a university hospital, a sample of 213 patients admitted to adult medical and surgical services was studied. Nineteen of these patients (9 percent) met the Diagnostic and Statistical Manual of Mental Disorders, ed 3, criteria for somatization disorder. A significantly higher proportion of the women compared with the men interviewed had the disorder (14 percent vs 3 percent, P less than .01). Fourteen percent of the divorced, separated, or widowed subjects qualified for the diagnosis, compared with 7 percent of the married subjects and 5 percent of the never-married subjects (P less than .05). Likewise, 32 percent of single patients with children at home had somatization disorder, compared with 4 percent of subjects in all other household configurations (P less than .001). When compared with matched controls, patients with somatization disorder had hospitalizations of roughly equal duration and expense, but had a much higher proportion of negative findings on workup for their presenting complaints (74 percent vs 21 percent, P less than .01). Nevertheless, these patients perceived their health as significantly worse than those without the disorder (P less than .001). None of the patients enrolled in this study had the diagnosis of somatization disorder at admission or discharge. This study documents that patients with somatization disorder are common, are unrecognized, and are admitted to the hospital for nonproductive workups. PMID:3681219

  19. Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula.

    PubMed

    Kurdyukov, Sergey; Song, Youhong; Tiew, Terence W-Y; Wang, Xin-Ding; Nolan, Kim E; Rose, Ray J

    2015-01-01

    Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by pre-globular, globular, heart, torpedo and cotyledon stages. This progressive development is under the tight regulation of a complex molecular network. Harvesting sufficient early embryos at a similar stage of development is essential for investigating the cellular and molecular regulation of early embryogenesis. This is not straightforward since early embryogenesis undergoes rapid morphogenesis in a short while e.g. 8 days for Medicago truncatula to reach the early cotyledon stage. Here, we address the issue by two approaches. The first one establishes a linkage between embryo development and pod morphology in helping indicate the stage of the zygotic embryo. This is particularly based on the number of pod spirals and development of the spines. An alternative way to complement the in vivo studies is via culturing leaf explants to produce somatic embryos. The medium includes an unusual hormone combination - an auxin (1-naphthaleneacetic acid), a cytokinin (6-benzylaminopurine), abscisic acid and gibberellic acid. The different stages can be discerned growing out of the callus without dissection. PMID:26131626

  20. Isolation and characterization of hardening-induced proteins in Chlorella vulgaris C-27: identification of late embryogenesis abundant proteins.

    PubMed

    Honjoh, K; Yoshimoto, M; Joh, T; Kajiwara, T; Miyamoto, T; Hatano, S

    1995-12-01

    Hardening-induced soluble proteins of Chlorella vulgaris Beijerink IAM C-27 (formerly Chlorella ellipsoidea Gerneck IAM C-27) were isolated and purified by two-dimensional high-performance liquid chromatography (2D-HPLC) on an anion-exchange column, with subsequent reversed-phase chromatography. Some of the proteins were resolved by SDS-PAGE, characterized by amino-terminal sequencing and identified by searching for homologies in databases. Separation of the soluble proteins during the hardening of Chlorella by a combination of 2D-HPLC and SDS-PAGE revealed that at least 31 proteins were induced or increased in abundance. Of particular interest was the induction after 12 h of a 10-kDa protein with the amino-terminal amino acid sequence AGNKPITEQISDAVGAAGQKVG and the induction after 6 h of a 14-kDa protein with the amino-terminal sequence ALGEESLGDKAKNAFEDAKDAVKDAAGNVKEAV. The amino-terminal sequences of these proteins indicated that they were homologous to late embryogenesis abundant (LEA) proteins. Furthermore, the level of a 22-kDa protein also increased after 12 h. The amino-terminal sequence of this protein, AAPLVGGPAPDFTAAAVFD, indicated that it was homologous to thioredoxin peroxidase. PMID:8589927

  1. Microspore embryogenesis and programmed cell death in barley: effects of copper on albinism in recalcitrant cultivars.

    PubMed

    Jacquard, Cédric; Nolin, Frédérique; Hécart, Carine; Grauda, Dace; Rashal, Isaak; Dhondt-Cordelier, Sandrine; Sangwan, Rajbir S; Devaux, Pierre; Mazeyrat-Gourbeyre, Florence; Clément, Christophe

    2009-09-01

    Albinism remains a major problem in cereal improvement programs that rely on doubled haploid (DH) technology, and the factors controlling the phenomenon are not well understood. Here we report on the positive influence of copper on the production of DH plants obtained through microspore embryogenesis (ME) in recalcitrant cultivars of barley (Hordeum vulgare L.). The presence of copper sulphate in the anther pre-treatment medium improved green DH plant regeneration from cultivars known to produce exclusively albino plants using classical procedures. In plastids, the effect of copper was characterized by a decrease in starch and a parallel increase in internal membranes. The addition of copper sulphate in the ME pre-treatment medium should enable breeders to exploit the genetic diversity of recalcitrant cultivars through DH technology. We examined programmed cell death (PCD) during microspore development to determine whether PCD may interfere with the induction of ME and/or the occurrence of albinism. By examining the fate of nuclei in various anther cell layers, we demonstrated that the kinetics of PCD in anthers differed between the barley cultivars Igri and Cork that show a low and a high rate of albinism, respectively. However, no direct correlation between PCD in the anther cell layers and the rate of albinism was observed and copper had no influence on the PCD kinetic in these cultivars. It was concluded that albinism following ME was not due to PCD in anthers, but rather to another unknown phenomenon that appears to specifically affect plastids during microspore/pollen development. PMID:19529940

  2. Identification of Late Embryogenesis Abundant (LEA) Protein Putative Interactors Using Phage Display

    PubMed Central

    Kushwaha, Rekha; Lloyd, Taylor D.; Schfermeyer, Kim R.; Kumar, Santosh; Downie, Allan Bruce

    2012-01-01

    Arabidopsis thaliana seeds without functional SEED MATURATION PROTEIN1 (SMP1), a boiling soluble protein predicted to be of intrinsic disorder, presumed to be a LATE EMBRYOGENESIS ABUNDANT (LEA) family protein based on sequence homology, do not enter secondary dormancy after 3 days at 40 C. We hypothesized that SMP1 may protect a heat labile protein involved in the promotion of secondary dormancy. Recombinant SMP1 and GmPM28, its soybean (Glycine max), LEA4 homologue, protected the labile GLUCOSE-6-PHOSPHATE DEHYROGENASE enzyme from heat stress, as did a known protectant, Bovine Serum Albumin, whether the LEA protein was in solution or attached to the bottom of microtiter plates. Maintenance of a biological function for both recombinant LEA proteins when immobilized encouraged a biopanning approach to screen for potential protein interactors. Phage display with two Arabidopsis seed, T7 phage, cDNA libraries, normalized for transcripts present in the mature, dehydrated, 12-, 24-, or 36-h imbibed seeds, were used in biopans against recombinant SMP1 and GmPM28. Phage titer increased considerably over four rounds of biopanning for both LEA proteins, but not for BSA, at both 25 and at 41 C, regardless of the library used. The prevalence of multiple, independent clones encoding portions of specific proteins repeatedly retrieved from different libraries, temperatures and baits, provides evidence suggesting these LEA proteins are discriminating which proteins they protect, a novel finding. The identification of putative LEA-interacting proteins provides targets for reverse genetic approaches to further dissect the induction of secondary dormancy in seeds in response to heat stress. PMID:22837651

  3. Effect of light conditions on anatomical and biochemical aspects of somatic and zygotic embryos of hybrid larch (Larix × marschlinsii)

    PubMed Central

    von Aderkas, Patrick; Teyssier, Caroline; Charpentier, Jean-Paul; Gutmann, Markus; Pâques, Luc; Le Metté, Claire; Ader, Kevin; Label, Philippe; Kong, Lisheng; Lelu-Walter, Marie-Anne

    2015-01-01

    Background and Aims In conifers, mature somatic embryos and zygotic embryos appear to resemble one another physiologically and morphologically. However, phenotypes of cloned conifer embryos can be strongly influenced by a number of in vitro factors and in some instances clonal variation can exceed that found in nature. This study examines whether zygotic embryos that develop within light-opaque cones differ from somatic embryos developing in dark/light conditions in vitro. Embryogenesis in larch is well understood both in situ and in vitro and thus provides a suitable system for addressing this question. Methods Features of somatic and zygotic embryos of hybrid larch, Larix × marschlinsii, were quantified, including cotyledon numbers, protein concentration and phenol chemistry. Somatic embryos were placed either in light or darkness for the entire maturation period. Embryos at different developmental stages were embedded and sectioned for histological analysis. Key Results Light, and to a lesser degree abscisic acid (ABA), influenced accumulation of protein and phenolic compounds in somatic and zygotic embryos. Dark-grown mature somatic embryos had more protein (91·77 ± 11·26 µg protein mg–1 f.wt) than either dark-grown zygotic embryos (62·40 ± 5·58) or light-grown somatic embryos (58·15 ± 10·02). Zygotic embryos never accumulated phenolic compounds at any stage, whereas somatic embryos stored phenolic compounds in the embryonal root caps and suspensors. Light induced the production of quercetrin (261·13 ± 9·2 µg g–1 d.wt) in somatic embryos. Mature zygotic embryos that were removed from seeds and placed on medium in light rapidly accumulated phenolics in the embryonal root cap and hypocotyl. Delaying germination with ABA delayed phenolic compound accumulation, restricting it to the embryonal root cap. Conclusions In larch embryos, light has a negative effect on protein accumulation, but a positive effect on phenol accumulation. Light did not affect morphogenesis, e.g. cotyledon number. Somatic embryos produced different amounts of phenolics, such as quercetrin, depending on light conditions. The greatest difference was seen in the embryonal root cap in all embryo types and conditions. PMID:25605662

  4. Contrasting Storage Protein Synthesis and Messenger RNA Accumulation during Development of Zygotic and Somatic Embryos of Alfalfa (Medicago sativa L.).

    PubMed

    Krochko, J E; Pramanik, S K; Bewley, J D

    1992-05-01

    During development on hormone-free media, somatic embryos pass through distinct morphological stages that superficially resemble those of zygotic embryo development (globular, heart, torpedo, cotyledonary stages). Despite these similarities, they differ from zygotic embryos in the extent of cotyledonary development and the patterns of synthesis and quantitative expression of seed-specific storage proteins (7S, 11S, and 2S proteins). Alfin (7S) is the first storage protein synthesized in developing zygotic embryos (stage IV). The 11S (medicagin) and 2S (Low Molecular Weight, LMW) storage proteins are not detectable until the following stage of development (stage V), although all three are present before the completion of embryo enlargement. Likewise, the 7S storage protein is the first to be synthesized in developing somatic embryos (day 5). Medicagin is evident by day 7 and the LMW protein by day 10. In contrast to zygotic embryos, alfin remains the predominant storage protein in somatic embryos throughout development. Not only are the relative amounts of medicagin and the LMW protein reduced in somatic embryos but the LMW protein is accumulated much later than the other proteins. Quantification of the storage protein mRNAs (7S, 11S, and 2S) by northern blot analysis confirms that there are substantial differences in the patterns of message accumulation in zygotic and somatic embryos of alfalfa (Medicago sativa). In zygotic embryos, the 7S, 11S, and 2S storage protein mRNAs are abundant during maturation and, in particular, during the stages of maximum protein synthesis (alfin, stages VI and VII; medicagin, stage VII; LMW, stage VII). In somatic embryos, the predominance of the 7S storage protein is correlated with increased accumulation of its mRNA, whereas the limited synthesis of the 11S storage protein is associated with much lower steady-state levels of its message. The mRNA for the LMW protein is present already by 3 days after transfer to hormone-free media, yet that protein is not evident on stained gels until day 10. Thus, both transcriptional and posttranscriptional events appear to be important in determining the protein complement of these seed tissues. On the basis of storage protein and mRNA accumulation, mature (14 days) somatic embryos most closely resemble stage VI zygotic embryos. The results of the developmental comparison also suggest that the patterns of synthesis of the individual storage proteins (7S, 11S, or 2S) are regulated independently of each other during embryogenesis in alfalfa. PMID:16668882

  5. Somatic Mutations in Cerebral Cortical Malformations

    PubMed Central

    Jamuar, Saumya S.; Lam, Anh-Thu N.; Kircher, Martin; D'Gama, Alissa M.; Wang, Jian; Barry, Brenda J.; Zhang, Xiaochang; Hill, Robert Sean; Partlow, Jennifer N.; Rozzo, Aldo; Servattalab, Sarah; Mehta, Bhaven K.; Topcu, Meral; Amrom, Dina; Andermann, Eva; Dan, Bernard; Parrini, Elena; Guerrini, Renzo; Scheffer, Ingrid E.; Berkovic, Samuel F.; Leventer, Richard J.; Shen, Yiping; Wu, Bai Lin; Barkovich, A. James; Sahin, Mustafa; Chang, Bernard S.; Bamshad, Michael; Nickerson, Deborah A.; Shendure, Jay; Poduri, Annapurna; Yu, Timothy W.; Walsh, Christopher A.

    2014-01-01

    BACKGROUND Although there is increasing recognition of the role of somatic mutations in genetic disorders, the prevalence of somatic mutations in neurodevelopmental disease and the optimal techniques to detect somatic mosaicism have not been systematically evaluated. METHODS Using a customized panel of known and candidate genes associated with brain malformations, we applied targeted high-coverage sequencing (depth, ≥200×) to leukocyte-derived DNA samples from 158 persons with brain malformations, including the double-cortex syndrome (subcortical band heterotopia, 30 persons), polymicrogyria with megalencephaly (20), periventricular nodular heterotopia (61), and pachygyria (47). We validated candidate mutations with the use of Sanger sequencing and, for variants present at unequal read depths, subcloning followed by colony sequencing. RESULTS Validated, causal mutations were found in 27 persons (17%; range, 10 to 30% for each phenotype). Mutations were somatic in 8 of the 27 (30%), predominantly in persons with the double-cortex syndrome (in whom we found mutations in DCX and LIS1), persons with periventricular nodular heterotopia (FLNA), and persons with pachygyria (TUBB2B). Of the somatic mutations we detected, 5 (63%) were undetectable with the use of traditional Sanger sequencing but were validated through subcloning and subsequent sequencing of the subcloned DNA. We found potentially causal mutations in the candidate genes DYNC1H1, KIF5C, and other kinesin genes in persons with pachygyria. CONCLUSIONS Targeted sequencing was found to be useful for detecting somatic mutations in patients with brain malformations. High-coverage sequencing panels provide an important complement to whole-exome and whole-genome sequencing in the evaluation of somatic mutations in neuropsychiatric disease. (Funded by the National Institute of Neurological Disorders and Stroke and others.) PMID:25140959

  6. Polyamine and Its Metabolite H2O2 Play a Key Role in the Conversion of Embryogenic Callus into Somatic Embryos in Upland Cotton (Gossypium hirsutum L.)

    PubMed Central

    Cheng, Wen-Han; Wang, Fan-Long; Cheng, Xin-Qi; Zhu, Qian-Hao; Sun, Yu-Qiang; Zhu, Hua-Guo; Sun, Jie

    2015-01-01

    The objective of this study was to increase understanding about the mechanism by which polyamines (PAs) promote the conversion of embryogenic calli (EC) into somatic embryos in cotton (Gossypium hirsutum L.). We measured the levels of endogenous PAs and H2O2, quantified the expression levels of genes involved in the PAs pathway at various stages of cotton somatic embryogenesis (SE), and investigated the effects of exogenous PAs and H2O2 on differentiation and development of EC. Putrescine (Put), spermidine (Spd), and spermine (Spm) significantly increased from the EC stage to the early phase of embryo differentiation. The levels of Put then decreased until the somatic embryo stage whereas Spd and Spm remained nearly the same. The expression profiles of GhADC genes were consistent with changes in Put during cotton SE. The H2O2 concentrations began to increase significantly at the EC stage, during which time both GhPAO1 and GhPAO4 expressions were highest and PAO activity was significantly increased. Exogenous Put, Spd, Spm, and H2O2 not only enhanced embryogenic callus growth and embryo formation, but also alleviated the effects of D-arginine and 1, 8-diamino-octane, which are inhibitors of PA synthesis and PAO activity. Overall, the results suggest that both PAs and their metabolic product H2O2 are essential for the conversion of EC into somatic embryos in cotton. PMID:26697030

  7. Micropropagation of Elaeis guineensis Jacq. 'Dura': Comparison of three basal media for efficient regeneration.

    PubMed

    Muniran, F; Bhore, Subhash J; Shah, Farida H

    2008-01-01

    Three basal plant tissue culture media, namely, N6, MS, and modified Y3, were compared to optimize micropropagation protocol for E. guineensis. Full strength media were used separately to regenerate plantlets directly using immature zygotic embryos (IZEs), and through somatic embryogenesis of calli obtained from IZEs. The plantlets regenerated by direct regeneration on three media were examined for shoot length and rooting percentage. For the induction of callus, somatic embryogenesis, and rooting modified Y3 medium was the most effective. In conclusion, the results indicate that modified Y3 medium is the most suitable for direct regeneration, callus induction and somatic embryogenesis in E. guineensis. PMID:18697576

  8. Induction voidmeter

    DOEpatents

    Anderson, T.T.; Roop, C.J.; Schmidt, K.J.; Brewer, J.

    1983-12-21

    An induction voidmeter for detecting voids in a conductive fluid may comprise: a four arm bridge circuit having two adjustable circuit elements connected as opposite arms of said bridge, an input branch, and an output branch; two induction coils, bifilarly wound together, connected as the remaining two opposing arms of said bridge circuit and positioned such that the conductive fluid passes through said coils; means for applying an AC excitation signal to said input branch; and means for detecting the output signal generated in response to said excitation signal across said output branch. The induction coils may be located outside or inside a non-magnetic pipe containing the conductive fluid.

  9. Induction voidmeter

    DOEpatents

    Anderson, Thomas T.; Roop, Conard J.; Schmidt, Kenneth J.; Brewer, John

    1986-01-01

    An induction voidmeter for detecting voids in a conductive fluid may comprise: a four arm bridge circuit having two adjustable circuit elements connected as opposite arms of said bridge circuit, an input branch, and an output branch; two induction coils, bifilarly wound together, connected as the remaining two opposing arms of said bridge circuit and positioned such that the conductive fluid passes through said coils; applying an AC excitation signal to said input branch; and detecting the output signal generated in response to said excitation signal across said output branch. The induction coils may be located outside or inside a non-magnetic pipe containing the conductive fluid.

  10. Potassium and carrot embryogenesis: are K+ channels necessary for development?

    PubMed

    Costa, Alex; Carpaneto, Armando; Varotto, Serena; Formentin, Elide; Marin, Oriano; Barizza, Elisabetta; Terzi, Mario; Gambale, Franco; Lo Schiavo, Fiorella

    2004-04-01

    The expression pattern of the KDC1 gene, coding for an inwardly-rectifying K(+) channel of Daucus carota , is described in several embryo stages and seedling tissues. Relative quantitative RT-PCR experiments indicated that, during (somatic) embryonic development, the KDC1 transcript appears as early as the globular stage and that the transcript level remains constant throughout the successive heart and torpedo stages. Thereafter, the KDC1 transcript is preferentially expressed in plant roots, but is also present in other tissues, and in particular, in the shoot apical meristem. In situ hybridisation experiments showed that in embryos KDC1 mRNA is detectable preferentially in protoderm cells with a stage dependent expression pattern. At later times, the hybridisation signal is particularly evident in root hairs, root epidermis and endodermis, but is also observed in single cell layers corresponding to L1 of the shoot apical meristem and leaf primordia. Promoter studies with the beta -glucuronidase reporter gene confirm preferential expression of KDC1 in embryo protoderm cells and in plant root epidermis and root hairs. Western blot analysis of embryonic proteins and immunolocalisation experiments on somatic embryos sections revealed the presence of KDC1 during embryo development. Consistent with these observations, patch-clamp experiments performed on protoplasts isolated from embryos at the torpedo stage demonstrated the presence of functional inward rectifying K(+) channels. This is the first report on the expression of a plant ion channel during embryo development. PMID:15604655

  11. The effects of microgravity on gametogenesis, fertilization, and early embryogenesis

    NASA Astrophysics Data System (ADS)

    Tan, X.

    Gametogenesis fertilization and early embryogenesis are crucial periods for normal development afterwards In past three decades many experiments have been conducted in space and in simulated weightlessness induced by clinostats to elucidate the issue Different animal species including Drosophila wasp shrimp fish amphibian mouse rats etc have been used for the study Oogenesis and spermatogenesis are affected by microgravity in different ways Some researches found that microgravity condition perturbed the process of oogenesis in many species A significant increased frequency of chromosomal non-disjunction was found in Drosophila females resulting the loss of chromosomes during meiosis and inhibition of cell division Studies on wasp showed a decreased hatchability and accumulation of unhatched eggs when the insects were exposed to spaceflight at different stages of oogenesis For experiments conducted on vertebrate animal models the results are somehow different however Microgravity has no significant effect for fish Medaka etc amphibian South African clawed toad Xenopus laevis or mammals mouse Spermatogenesis on the other hand is more significantly affected by microgravity condition Some researches indicated sperm are sensitive to changes in gravitational force and this sensitivity affects the ability of sperm to fertilize eggs Sperm swim with higher velocity in microgravity which is coupled with altered protein phosphorylation level in sperm under microgravity condition Microgravity also induced activation of the

  12. The ETS transcription factor GABPalpha is essential for early embryogenesis.

    PubMed

    Ristevski, Sika; O'Leary, Debra A; Thornell, Anders P; Owen, Michael J; Kola, Ismail; Hertzog, Paul J

    2004-07-01

    The ETS transcription factor complex GABP consists of the GABPalpha protein, containing an ETS DNA binding domain, and an unrelated GABPbeta protein, containing a transactivation domain and nuclear localization signal. GABP has been shown in vitro to regulate the expression of nuclear genes involved in mitochondrial respiration and neuromuscular signaling. We investigated the in vivo function of GABP by generating a null mutation in the murine Gabpalpha gene. Embryos homozygous for the null Gabpalpha allele die prior to implantation, consistent with the broad expression of Gabpalpha throughout embryogenesis and in embryonic stem cells. Gabpalpha(+/-) mice demonstrated no detectable phenotype and unaltered protein levels in the panel of tissues examined. This indicates that Gabpalpha protein levels are tightly regulated to protect cells from the effects of loss of Gabp complex function. These results show that Gabpalpha function is essential and is not compensated for by other ETS transcription factors in the mouse, and they are consistent with a specific requirement for Gabp expression for the maintenance of target genes involved in essential mitochondrial cellular functions during early cleavage events of the embryo. PMID:15199140

  13. Expression of evolutionarily conserved eye specification genes during Drosophila embryogenesis

    PubMed Central

    Kumar, Justin P.; Moses, Kevin

    2009-01-01

    Eye specification in Drosophila is thought be controlled by a set of seven nuclear factors that includes the Pax6 homolog, Eyeless. This group of genes is conserved throughout evolution and has been repeatedly recruited for eye specification. Several of these genes are expressed within the developing eyes of vertebrates and mutations in several mouse and human orthologs are the underlying causes of retinal disease syndromes. Ectopic expression in Drosophila of any one of these genes is capable of inducing retinal development, while loss-of-function mutations delete the developing eye. These nuclear factors comprise a complex regulatory network and it is thought that their combined activities are required for the formation of the eye. We examined the expression patterns of four eye specification genes, eyeless (ey), sine oculis (so), eyes absent (eya), and dachshund (dac) throughout all time points of embryogenesis and show that only eyeless is expressed within the embryonic eye anlagen. This is consistent with a recently proposed model in which the eye primordium acquires its competence to become retinal tissue over several time points of development. We also compare the expression of Ey with that of a putative antennal specifying gene Distal-less (Dll). The expression patterns described here are quite intriguing and raise the possibility that these genes have even earlier and wide ranging roles in establishing the head and visual field. PMID:11685574

  14. Expression and cellular localization of Atrab28 during arabidopsis embryogenesis.

    PubMed

    Arenas-Mena, C; Raynal, M; Borrell, A; Varoquaux, F; Cutanda, M C; Stacy, R A; Pagès, M; Delseny, M; Culiáñez-Macià, F A

    1999-05-01

    The maize abscisic acid (ABA)-responsive gene rab28 has been shown to be ABA-inducible in embryos and vegetative tissues, expression being mostly restricted to vascular elements during late embryogenesis. In the course of an expressed sequence tags (ESTs) programme, we have isolated an Arabidopsis thaliana gene, Atrab28, encoding the orthologue of maize rab28. The Atrab28 cDNA is 1090 bp long, including a poly(A)+ stretch, and encodes a polypeptide of 262 amino acids. Atrab28 antibody against the recombinant protein recognizes a polipeptide of about 30 kDa and pI 6, in close agreement with the predicted molecular mass and pI. As for maize rab28, expression studies with Atrab28 revealed high specificity for embryo tissues, transcription being stimulated by the transcriptional activator abi3. In contrast, Atrab28 was not induced in vegetative tissues by ABA, osmotic stress or dehydration. The expression of Atrab28 mRNA and the accumulation of Atrab28 protein was largely restricted to provascular tissues of mature embryos and in the seed coat outer tegument and embryo and silique epidermis, as revealed by in situ hybridization and immunocytochemistry with anti-Atrab28 antibodies. PMID:10412913

  15. Embryogenesis, hatching and larval development of Artemia during orbital spaceflight

    NASA Astrophysics Data System (ADS)

    Spooner, B. S.; Debell, L.; Armbrust, L.; Guikema, J. A.; Metcalf, J.; Paulsen, A.

    1994-08-01

    Developmental biology studies, using gastrula-arrested cysts of the brine shrimp Artemia franciscana, were conducted during two flights of the space shuttle Atlantis (missions STS-37 and STS-43) in 1991. Dehydrated cysts were activated, on orbit, by addition of salt water to the cysts, and then development was terminated by the addition of fixative. Development took place in 5 ml syringes, connected by tubing to activation syringes, containing salt water, and termination syringes, containing fixative. Comparison of space results with simultaneous ground control experiments showed that equivalent percentages of naupliar larvae hatched in the syringes (40%). Thus, reactivation of development, completion of embryogenesis, emergence and hatching took place, during spaceflight, without recognizable alteration in numbers of larvae produced. Post-hatching larval development was studied in experiments where development was terminated, by intrduction of fixative, 2 days, 4 days, and 8 days after reinitiation of development. During spaceflight, successive larval instars or stages, interrupted by molts, occurred, generating brine shrimp at appropriate larval instars. Naupliar larvae possessed the single naupliar eye, and development of the lateral pair of adult eyes also took place in space. Transmission electron microscopy revealed extensive differentiation, including skeletal muscle and gut endoderm, as well as the eye tissues. These studies demonstrate the potential value of Artemia for developmental biology studies during spaceflight, and show that extensive degress of development can take place in this microgravity environment.

  16. Embryogenesis, hatching and larval development of Artemia during orbital spaceflight

    NASA Technical Reports Server (NTRS)

    Spooner, B. S.; Debell, L.; Armbrust, L.; Guikema, J. A.; Metcalf, J.; Paulsen, A.

    1994-01-01

    Developmental biology studies, using gastrula-arrested cysts of the brine shrimp Artemia franciscana, were conducted during two flights of the space shuttle Atlantis (missions STS-37 and STS-43) in 1991. Dehydrated cysts were activated, on orbit, by addition of salt water to the cysts, and then development was terminated by the addition of fixative. Development took place in 5 ml syringes, connected by tubing to activation syringes, containing salt water, and termination syringes, containing fixative. Comparison of space results with simultaneous ground control experiments showed that equivalent percentages of naupliar larvae hatched in the syringes (40%). Thus, reactivation of development, completion of embryogenesis, emergence and hatching took place, during spaceflight, without recognizable alteration in numbers of larvae produced. Post-hatching larval development was studied in experiments where development was terminated, by introduction of fixative, 2 days, 4 days, and 8 days after reinitiation of development. During spaceflight, successive larval instars or stages, interrupted by molts, occurred, generating brine shrimp at appropriate larval instars. Naupliar larvae possessed the single naupliar eye, and development of the lateral pair of adult eyes also took place in space. Transmission electron microscopy revealed extensive differentiation, including skeletal muscle and gut endoderm, as well as the eye tissues. These studies demonstrate the potential value of Artemia for developmental biology studies during spa ceflight, and show that extensive degrees of development can take place in this microgravity environment.

  17. Modeling cell-cycle synchronization during embryogenesis in Xenopus laevis

    NASA Astrophysics Data System (ADS)

    McIsaac, R. Scott; Huang, K. C.; Sengupta, Anirvan; Wingreen, Ned

    2010-03-01

    A widely conserved aspect of embryogenesis is the ability to synchronize nuclear divisions post-fertilization. How is synchronization achieved? Given a typical protein diffusion constant of 10 μm^2sec, and an embryo length of 1mm, it would take diffusion many hours to propagate a signal across the embryo. Therefore, synchrony cannot be attained by diffusion alone. We hypothesize that known autocatalytic reactions of cell-cycle components make the embryo an ``active medium'' in which waves propagate much faster than diffusion, enforcing synchrony. We report on robust spatial synchronization of components of the core cell cycle circuit based on a mathematical model previously determined by in vitro experiments. In vivo, synchronized divisions are preceded by a rapid calcium wave that sweeps across the embryo. Experimental evidence supports the hypothesis that increases in transient calcium levels lead to derepression of a negative feedback loop, allowing cell divisions to start. Preliminary results indicate a novel relationship between the speed of the initial calcium wave and the ability to achieve synchronous cell divisions.

  18. Toward establishing a morphological and ultrastructural characterization of proembryogenic masses and early somatic embryos of Araucaria angustifolia (Bert.) O. Kuntze.

    PubMed

    Steiner, Neusa; Farias-Soares, Francine L; Schmidt, Éder C; Pereira, Maria L T; Scheid, Bruna; Rogge-Renner, Gladys D; Bouzon, Zenilda L; Schmidt, Daniela; Maldonado, Sara; Guerra, Miguel P

    2016-03-01

    Somatic embryogenesis is a morphogenetic route useful for the study of embryonic development, as well as the large-scale propagation of endangered species, such as the Brazilian pine (Araucaria angustifolia). In the present study, we investigated the morphological and ultrastructural organization of A. angustifolia somatic embryo development by means of optical and electron microscopy. The proembryogenic stage was characterized by the proliferation of proembryogenic masses (PEMs), which are cellular aggregates composed of embryogenic cells (ECs) attached to suspensor-like cells (SCs). PEMs proliferate through three developmental stages, PEM I, II, and III, by changes in the number of ECs and SCs. PEM III-to-early somatic embryo (SE) transition was characterized by compact clusters of ECs growing out of PEM III, albeit still connected to it by SCs. Early SEs showed a dense globular embryonic mass (EM) and suspensor region (SR) connected by embryonic tube cells (TCs). By comparison, early somatic and zygotic embryos showed similar morphology. ECs are round with a large nucleus, nucleoli, and many cytoplasmic organelles. In contrast, TCs and SCs are elongated and vacuolated with cellular dismantling which is associated with programmed cell death of SCs. Abundant starch grains were observed in the TCs and SCs, while proteins were more abundant in the ECs. Based on the results of this study, a fate map of SE development in A. angustifolia is, for the first time, proposed. Additionally, this study shows the cell biology of SE development of this primitive gymnosperm which may be useful in evolutionary studies in this area. PMID:25968333

  19. Labor Induction

    MedlinePlus

    ... on labor? • When is amniotomy done? • What is oxytocin? • What are the risks associated with labor induction? • ... amniotic sac breaks (their “water breaks”). What is oxytocin? Oxytocin is a hormone that causes contractions of ...

  20. Cloning by somatic cell nuclear transfer.

    PubMed

    Fulka, J; First, N L; Loi, P; Moor, R M

    1998-10-01

    The birth of the first cloned mammals, produced by the introduction of somatic cell nuclei into enucleated oocytes, was an impressive and surprising development. Although the ethical debate has been intense, the important scientific questions raised by this work have been inadequately discussed and are still unresolved. In this essay we address three questions about nuclear transplantation in the eggs of mice and domestic animals. First, why were the recent experiments on somatic cell cloning successful, when so many others have failed? Second, were these exceptional cases, or is somatic cloning now open to all? Third, what are the future possibilities for increasing the efficiency and wider applicability of the cloning process? PMID:9819572

  1. Landscape of Somatic Retrotransposition in Human Cancers

    PubMed Central

    Lee, Eunjung; Iskow, Rebecca; Yang, Lixing; Gokcumen, Omer; Haseley, Psalm; Luquette, Lovelace J.; Lohr, Jens G.; Harris, Christopher C.; Ding, Li; Wilson, Richard K.; Wheeler, David A.; Gibbs, Richard A.; Kucherlapati, Raju; Lee, Charles; Kharchenko, Peter V.; Park, Peter J.

    2013-01-01

    Transposable elements (TEs) are abundant in the human genome, and some are capable of generating new insertions through RNA intermediates. In cancer, the disruption of cellular mechanisms that normally suppress TE activity may facilitate mutagenic retrotranspositions. We performed single-nucleotide resolution analysis of TE insertions in 43 high-coverage whole-genome sequencing data sets from five cancer types. We identified 194 high-confidence somatic TE insertions, as well as thousands of polymorphic TE insertions in matched normal genomes. Somatic insertions were present in epithelial tumors but not in blood or brain cancers. Somatic L1 insertions tend to occur in genes that are commonly mutated in cancer, disrupt the expression of the target genes, and are biased toward regions of cancer-specific DNA hypomethylation, highlighting their potential impact in tumorigenesis. PMID:22745252

  2. Coherent Somatic Mutation in Autoimmune Disease

    PubMed Central

    Ross, Kenneth Andrew

    2014-01-01

    Background Many aspects of autoimmune disease are not well understood, including the specificities of autoimmune targets, and patterns of co-morbidity and cross-heritability across diseases. Prior work has provided evidence that somatic mutation caused by gene conversion and deletion at segmentally duplicated loci is relevant to several diseases. Simple tandem repeat (STR) sequence is highly mutable, both somatically and in the germ-line, and somatic STR mutations are observed under inflammation. Results Protein-coding genes spanning STRs having markers of mutability, including germ-line variability, high total length, repeat count and/or repeat similarity, are evaluated in the context of autoimmunity. For the initiation of autoimmune disease, antigens whose autoantibodies are the first observed in a disease, termed primary autoantigens, are informative. Three primary autoantigens, thyroid peroxidase (TPO), phogrin (PTPRN2) and filaggrin (FLG), include STRs that are among the eleven longest STRs spanned by protein-coding genes. This association of primary autoantigens with long STR sequence is highly significant (). Long STRs occur within twenty genes that are associated with sixteen common autoimmune diseases and atherosclerosis. The repeat within the TTC34 gene is an outlier in terms of length and a link with systemic lupus erythematosus is proposed. Conclusions The results support the hypothesis that many autoimmune diseases are triggered by immune responses to proteins whose DNA sequence mutates somatically in a coherent, consistent fashion. Other autoimmune diseases may be caused by coherent somatic mutations in immune cells. The coherent somatic mutation hypothesis has the potential to be a comprehensive explanation for the initiation of many autoimmune diseases. PMID:24988487

  3. Animal cloning by somatic cell nuclear transfer.

    PubMed

    Smith, Lawrence C; Yoo, Jae-Gyu

    2009-01-01

    Animal cloning is becoming increasingly useful for its applications in biological inquiry and for its potential use in pharmaceutical, medical, and agricultural fields. Due to the complexity of the numerous steps required in reconstructing oocytes by nuclear transfer, detailed protocols are required to minimize the developmental damages inflicted during these manipulations and to standardize procedures across laboratories. Moreover, because oogenesis and early embryogenesis differ widely among mammalian species, it is essential that protocols be adapted according to each species concerned. Our objective here is to detail the protocols that have been most successful in producing laboratory and domestic animal clones. PMID:19495710

  4. Neuron navigator 3a regulates liver organogenesis during zebrafish embryogenesis.

    PubMed

    Klein, Christian; Mikutta, Janine; Krueger, Janna; Scholz, Katja; Brinkmann, Joep; Liu, Dong; Veerkamp, Justus; Siegel, Doreen; Abdelilah-Seyfried, Salim; le Noble, Ferdinand

    2011-05-01

    Endodermal organogenesis requires a precise orchestration of cell fate specification and cell movements, collectively coordinating organ size and shape. In Caenorhabditis elegans, uncoordinated-53 (unc-53) encodes a neural guidance molecule that directs axonal growth. One of the vertebrate homologs of unc-53 is neuron navigator 3 (Nav3). Here, we identified a novel vertebrate neuron navigator 3 isoform in zebrafish, nav3a, and we provide genetic evidence in loss- and gain-of-function experiments showing its functional role in endodermal organogenesis during zebrafish embryogenesis. In zebrafish embryos, nav3a expression was initiated at 22 hpf in the gut endoderm and at 40 hpf expanded to the newly formed liver bud. Endodermal nav3a expression was controlled by Wnt2bb signaling and was independent of FGF and BMP signaling. Morpholino-mediated knockdown of nav3a resulted in a significantly reduced liver size, and impaired development of pancreas and swim bladder. In vivo time-lapse imaging of liver development in nav3a morphants revealed a failure of hepatoblast movement out from the gut endoderm during the liver budding stage, with hepatoblasts being retained in the intestinal endoderm. In hepatocytes in vitro, nav3a acts as a positive modulator of actin assembly in lamellipodia and filipodia extensions, allowing cellular movement. Knockdown of nav3a in vitro impeded hepatocyte movement. Endodermal-specific overexpression of nav3a in vivo resulted in additional ectopic endodermal budding beyond the normal liver and pancreatic budding sites. We conclude that nav3a is required for directing endodermal organogenesis involving coordination of endodermal cell behavior. PMID:21471154

  5. Alterations in chromatin structure during early sea urchin embryogenesis.

    PubMed Central

    Savić, A; Richman, P; Williamson, P; Poccia, D

    1981-01-01

    Sea urchin sperm before fertilization possess the longest nucleosome repeat length yet determined for any chromatin. By the time the fertilized egg gives rise to a blastula or gastrula embryo, the chromatin has a considerably shorter repeat length and, in addition, a sequence of different histone variants of H1, H2A, and H2B has appeared. We have investigated the relationship between these variations in histone composition and concomitant alterations in chromatin structure during the earliest stages of embryogenesis in two species of sea urchin. In contrast to the long repeat distance in sperm, chromatin loaded with cleavage stage histones has a much smaller repeat. Later stages containing predominantly alpha histones display an intermediate spacing. More detailed analysis of the events in the first cell cycle was carried out with polyspermically fertilized eggs. During the first 30 min after fertilization, in which sperm-specific H1 is completely replaced by cleavage-stage H1, the male pronuclear repeat remains unchanged. The decrease toward the repeat length of cleavage stages begins at about the time of DNA synthesis. Higher degrees of polyspermy extend the length of the cell cycle, including the duration of S phase and the length of time to reach the first chromosome condensation. At these higher degrees of polyspermy, the decrease in repeat length is also slowed. We conclude that the adjustment of the arrangement of nucleosomes in embryonic chromatin from that found in sperm can occur within the first cell cycle and that its timing is cell-cycle dependent. The adjustment is separable from a corresponding change in H1 composition. Images PMID:6943576

  6. Indispensable Roles of Plastids in Arabidopsis thaliana Embryogenesis

    PubMed Central

    Hsu, Shih-Chi; Belmonte, Mark F; Harada, John J; Inoue, Kentaro

    2010-01-01

    The plastid is an organelle vital to all photosynthetic and some non-photosynthetic eukaryotes. In the model plant Arabidopsis thaliana, a number of nuclear genes encoding plastid proteins have been found to be necessary for embryo development. However, the exact roles of plastids in this process remain largely unknown. Here we use publicly available datasets to obtain insights into the relevance of plastid activities to A. thaliana embryogenesis. By searching the SeedGenes database (http://www.seedgenes.org) and recent literature, we found that, of the 339 non-redundant genes required for proper embryo formation, 108 genes likely encode plastid-targeted proteins. Nineteen of these genes are necessary for development of preglobular embryos and/or their conversion to globular embryos, of which 13 genes encode proteins involved in non-photosynthetic metabolism. By contrast, among 38 genes which are dispensable for globular embryo formation but necessary for further development, only one codes for a protein involved in metabolism. Products of 21 of the 38 genes play roles in plastid gene expression and maintenance. Examination of RNA profiles of embryos at distinct growth stages obtained in laser-capture microdissection coupled with DNA microarray experiments revealed that most of the identified genes are expressed throughout embryo morphogenesis and maturation. These findings suggest that metabolic activities are required at preglobular and throughout all stages of embryo development, whereas plastid gene expression becomes necessary during and/or after the globular stage to sustain various activities of the organelle including photosynthetic electron transport. PMID:21286311

  7. Exploring Psychophysiological Markers of Vulnerability to Somatic Illnesses in Females

    PubMed Central

    Keenan, Kate; Marsland, Anna

    2009-01-01

    Objective To examine the association between biological stress regulation and somatic complaints in young girls prior to the onset of clear psychopathology such as somatization disorder. Methods Salivary cortisol, heart rate variability (HRV), and negative mood were assessed in 48 12-year-old girls in response to the Trier Social Stress Test for Children (TSST-C). Parent and child report on the Children's Somatization Inventory was used to identify girls with high and low somatic complaints. Results Girls with high levels of somatic complaints had significantly higher initial levels of cortisol, which decreased over time, and showed a trend for a more limited HRV in response to the TSST-C than girls with low levels of somatic complaints. Conclusions High levels of cortisol and possibly low HRV among girls with somatic complaints may interfere with flexibility in responding to typical psychosocial stressors, which may increase vulnerability to the onset of somatic illnesses in females. PMID:19286887

  8. Checkpoint kinase 1 negatively regulates somatic hypermutation.

    PubMed

    Frankenberger, Samantha; Davari, Kathrin; Fischer-Burkart, Sabine; Böttcher, Katrin; Tomi, Nils-Sebastian; Zimber-Strobl, Ursula; Jungnickel, Berit

    2014-04-01

    Immunoglobulin (Ig) diversification by somatic hypermutation in germinal center B cells is instrumental for maturation of the humoral immune response, but also bears the risk of excessive or aberrant genetic changes. Thus, introduction of DNA damage by activation-induced cytidine deaminase as well as DNA repair by multiple pathways need to be tightly regulated during the germinal center response to prevent lymphomagenesis. In the present study, we show that DNA damage checkpoint signaling via checkpoint kinase 1 (Chk1) negatively regulates somatic hypermutation. Chk1 inhibition in human B cell lymphoma lines as well as inactivation of Chk1 alleles by gene targeting in DT40 B cells leads to increased somatic hypermutation. This is apparently due to changes in DNA repair pathways regulated by Chk1, such as a decreased homologous recombination efficiency that also leads to decreased Ig gene conversion in DT40. Our data show that Chk1 signaling plays a crucial role in regulation of Ig diversification and sheds unexpected light on potential origins of aberrant somatic hypermutation in B cell lymphomagenesis. PMID:24423870

  9. Checkpoint kinase 1 negatively regulates somatic hypermutation

    PubMed Central

    Frankenberger, Samantha; Davari, Kathrin; Fischer-Burkart, Sabine; Bttcher, Katrin; Tomi, Nils-Sebastian; Zimber-Strobl, Ursula; Jungnickel, Berit

    2014-01-01

    Immunoglobulin (Ig) diversification by somatic hypermutation in germinal center B cells is instrumental for maturation of the humoral immune response, but also bears the risk of excessive or aberrant genetic changes. Thus, introduction of DNA damage by activation-induced cytidine deaminase as well as DNA repair by multiple pathways need to be tightly regulated during the germinal center response to prevent lymphomagenesis. In the present study, we show that DNA damage checkpoint signaling via checkpoint kinase 1 (Chk1) negatively regulates somatic hypermutation. Chk1 inhibition in human B cell lymphoma lines as well as inactivation of Chk1 alleles by gene targeting in DT40 B cells leads to increased somatic hypermutation. This is apparently due to changes in DNA repair pathways regulated by Chk1, such as a decreased homologous recombination efficiency that also leads to decreased Ig gene conversion in DT40. Our data show that Chk1 signaling plays a crucial role in regulation of Ig diversification and sheds unexpected light on potential origins of aberrant somatic hypermutation in B cell lymphomagenesis. PMID:24423870

  10. Somatic Symptoms in Traumatized Children and Adolescents

    ERIC Educational Resources Information Center

    Kugler, Brittany B.; Bloom, Marlene; Kaercher, Lauren B.; Truax, Tatyana V.; Storch, Eric A.

    2012-01-01

    Childhood exposure to trauma has been associated with increased rates of somatic symptoms (SS), which may contribute to diminished daily functioning. One hundred and sixty-one children residing at a residential treatment home who had experienced neglect and/or abuse were administered the Trauma Symptom Checklist for Children (TSCC), the…

  11. Cloning and expression of embryogenesis-regulating genes in Araucaria angustifolia (Bert.) O. Kuntze (Brazilian Pine).

    PubMed

    Schlögl, Paulo Sérgio; Dos Santos, André Luis Wendt; Vieira, Leila do Nascimento; Floh, Eny Iochevet Segal; Guerra, Miguel Pedro

    2012-01-01

    Angiosperm and gymnosperm plants evolved from a common ancestor about 300 million years ago. Apart from morphological and structural differences in embryogenesis and seed origin, a set of embryogenesis-regulating genes and the molecular mechanisms involved in embryo development seem to have been conserved alike in both taxa. Few studies have covered molecular aspects of embryogenesis in the Brazilian pine, the only economically important native conifer in Brazil. Thus eight embryogenesis-regulating genes, viz., ARGONAUTE 1, CUP-SHAPED COTYLEDON 1, WUSCHEL-related WOX, S-LOCUS LECTIN PROTEIN KINASE, SCARECROW-like, VICILIN 7S, LEAFY COTYLEDON 1, and REVERSIBLE GLYCOSYLATED POLYPEPTIDE 1, were analyzed through semi-quantitative RT-PCR during embryo development and germination. All the eight were found to be differentially expressed in the various developmental stages of zygotic embryos, seeds and seedling tissues. To our knowledge, this is the first report on embryogenesis-regulating gene expression in members of the Araucariaceae family, as well as in plants with recalcitrant seeds. PMID:22481892

  12. An optimized procedure for plant recovery from somatic embryos significantly facilitates the genetic improvement of Vitis.

    PubMed

    Li, Zhijian T; Kim, Kyung-Hee; Dhekney, Sadanand A; Jasinski, Jonathan R; Creech, Matthew R; Gray, Dennis J

    2014-01-01

    Plant regeneration from grapevine (Vitis spp.) via somatic embryogenesis typically is poor. Recovery of plants from Vitis rotundifolia Michx. (muscadine grape) is particularly problematic due to extremely low efficiency, including extended culture durations required for embryo-plant conversion. Poor plant recovery is an obstacle to the selection of improved genetically modified lines. Somatic embryos (SEs) of V. rotundifolia cultivar Delicious (Del-HS) and Vitis vinifera L cultivar Thompson Seedless (TS) were used to identify culture media and conditions that promoted embryo differentiation and plant conversion; this resulted in a two-step culture system. In comparative culture experiments, C2D medium containing 6% sucrose was the most effective, among four distinct formulae tested, for inducing precocious SE germination and cell differentiation. This medium, further supplemented with 4 µM 6-benzylaminopurine (C2D4B), was subsequently determined to enhance post-germinative growth of SE. MS medium supplemented with 0.5 µM 1-naphthaleneacetic acid (MSN) was then utilized to stimulate root and shoot growth of germinated SE. An average of 35% and 80% 'Del-HS' and 'TS' SE, respectively, developed into plants. All plants developed robust root and shoot systems and exhibited excellent survival following transfer to soil. Over 150 plants of 'Del-HS' were regenerated and established within 2.5 months, which is a dramatic reduction from the 6- to 12-month time period previously required. Similarly, 88 'TS' plant lines were obtained within the same time period. Subsequently, seven out of eight Vitis cultivars exhibited significantly increased plant conversion percentages, demonstrating broad application of the two-step culture system to produce the large numbers of independent plant lines needed for selection of desired traits. PMID:26504540

  13. An optimized procedure for plant recovery from somatic embryos significantly facilitates the genetic improvement of Vitis

    PubMed Central

    Li, Zhijian T; Kim, Kyung-Hee; Dhekney, Sadanand A; Jasinski, Jonathan R; Creech, Matthew R; Gray, Dennis J

    2014-01-01

    Plant regeneration from grapevine (Vitis spp.) via somatic embryogenesis typically is poor. Recovery of plants from Vitis rotundifolia Michx. (muscadine grape) is particularly problematic due to extremely low efficiency, including extended culture durations required for embryo–plant conversion. Poor plant recovery is an obstacle to the selection of improved genetically modified lines. Somatic embryos (SEs) of V. rotundifolia cultivar Delicious (Del-HS) and Vitis vinifera L cultivar Thompson Seedless (TS) were used to identify culture media and conditions that promoted embryo differentiation and plant conversion; this resulted in a two-step culture system. In comparative culture experiments, C2D medium containing 6% sucrose was the most effective, among four distinct formulae tested, for inducing precocious SE germination and cell differentiation. This medium, further supplemented with 4 µM 6-benzylaminopurine (C2D4B), was subsequently determined to enhance post-germinative growth of SE. MS medium supplemented with 0.5 µM 1-naphthaleneacetic acid (MSN) was then utilized to stimulate root and shoot growth of germinated SE. An average of 35% and 80% ‘Del-HS’ and ‘TS’ SE, respectively, developed into plants. All plants developed robust root and shoot systems and exhibited excellent survival following transfer to soil. Over 150 plants of ‘Del-HS’ were regenerated and established within 2.5 months, which is a dramatic reduction from the 6- to 12-month time period previously required. Similarly, 88 ‘TS’ plant lines were obtained within the same time period. Subsequently, seven out of eight Vitis cultivars exhibited significantly increased plant conversion percentages, demonstrating broad application of the two-step culture system to produce the large numbers of independent plant lines needed for selection of desired traits. PMID:26504540

  14. piggyBac transposon somatic mutagenesis with an activated reporter and tracker (PB-SMART) for genetic screens in mice.

    PubMed

    Landrette, Sean F; Cornett, Jonathan C; Ni, Thomas K; Bosenberg, Marcus W; Xu, Tian

    2011-01-01

    Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal. Retroviral and transposon mutagenesis systems in mice have been designed and deployed in somatic tissues for surveying hematopoietic and solid tumor formation. In the context of cancer, the ability to visually mark mutant cells would present tremendous advantages for identifying tumor formation, monitoring tumor growth over time, and tracking tumor infiltrations and metastases into wild-type tissues. Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes. For this purpose, we developed a system using the piggyBac (PB) transposon for somatic mutagenesis with an activated reporter and tracker, called PB-SMART. The PB-SMART mouse genetic screening system can simultaneously induce somatic mutations and mark mutated cells using bioluminescence or fluorescence. The marking of mutant cells enable analyses that are not possible with current somatic mutagenesis systems, such as tracking cell proliferation and tumor growth, detecting tumor cell infiltrations, and reporting tissue mutagenesis levels by a simple ex vivo visual readout. We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions. We further integrated a conditional transposase with the PB-SMART system, permitting tissue-specific mutagenesis with a single cross to any available Cre line. Targeting the germline, the system could also be used to conduct F1 screens. With these features, PB-SMART provides an integrated platform for individual investigators to harness the power of somatic mutagenesis and phenotypic screens to decipher the genetic basis of mammalian biology and disease. PMID:22039523

  15. Morphological abnormalities, impaired fetal development and decrease in myostatin expression following somatic cell nuclear transfer in dogs.

    PubMed

    Hong, Il-Hwa; Jeong, Yeon-Woo; Shin, Taeyoung; Hyun, Sang-Hwan; Park, Jin-Kyu; Ki, Mi-Ran; Han, Seon-Young; Park, Se-Il; Lee, Ji-Hyun; Lee, Eun-Mi; Kim, Ah-Young; You, Sang-Young; Hwang, Woo-Suk; Jeong, Kyu-Shik

    2011-05-01

    Several mammals, including dogs, have been successfully cloned using somatic cell nuclear transfer (SCNT), but the efficiency of generating normal, live offspring is relatively low. Although the high failure rate has been attributed to incomplete reprogramming of the somatic nuclei during the cloning process, the exact cause is not fully known. To elucidate the cause of death in cloned offspring, 12 deceased offspring cloned by SCNT were necropsied. The clones were either stillborn just prior to delivery or died with dyspnea shortly after birth. On gross examination, defects in the anterior abdominal wall and increased heart and liver sizes were found. Notably, a significant increase in muscle mass and macroglossia lesions were observed in deceased SCNT-cloned dogs. Interestingly, the expression of myostatin, a negative regulator of muscle growth during embryogenesis, was down-regulated at the mRNA level in tongues and skeletal muscles of SCNT-cloned dogs compared with a normal dog. Results of the present study suggest that decreased expression of myostatin in SCNT-cloned dogs may be involved in morphological abnormalities such as increased muscle mass and macroglossia, which may contribute to impaired fetal development and poor survival rates. PMID:21520324

  16. Chromatin dynamics in pollen mother cells underpin a common scenario at the somatic-to-reproductive fate transition of both the male and female lineages in Arabidopsis

    PubMed Central

    She, Wenjing; Baroux, Célia

    2015-01-01

    Unlike animals, where the germline is established early during embryogenesis, plants set aside their reproductive lineage late in development in dedicated floral organs. The specification of pollen mother cells (PMC) committed to meiosis takes place in the sporogenous tissue in anther locules and marks the somatic-to-reproductive cell fate transition toward the male reproductive lineage. Here we show that Arabidopsis PMC differentiation is accompanied by large-scale changes in chromatin organization. This is characterized by significant increase in nuclear volume, chromatin decondensation, reduction in heterochromatin, eviction of linker histones and the H2AZ histone variant. These structural alterations are accompanied by dramatic, quantitative changes in histone modifications levels compared to that of surrounding somatic cells that do not share a sporogenic fate. All these changes are highly reminiscent of those we have formerly described in female megaspore mother cells (MMC). This indicates that chromatin reprogramming is a common underlying scenario in the somatic-to-reproductive cell fate transition in both male and female lineages. PMID:25972887

  17. Emerging patterns of somatic mutations in cancer

    PubMed Central

    Watson, Ian R.; Takahashi, Koichi; Futreal, P. Andrew; Chin, Lynda

    2014-01-01

    The advance in technological tools for massively parallel, high-throughput sequencing of DNA has enabled the comprehensive characterization of somatic mutations in large number of tumor samples. Here, we review recent cancer genomic studies that have assembled emerging views of the landscapes of somatic mutations through deep sequencing analyses of the coding exomes and whole genomes in various cancer types. We discuss the comparative genomics of different cancers, including mutation rates, spectrums, and roles of environmental insults that influence these processes. We highlight the developing statistical approaches used to identify significantly mutated genes, and discuss the emerging biological and clinical insights from such analyses as well as the challenges ahead translating these genomic data into clinical impacts. PMID:24022702

  18. Somatic Cell Reprogramming into Cardiovascular Lineages

    PubMed Central

    Chen, Jenny X.; Plonowska, Karolina; Wu, Sean M.

    2015-01-01

    Ischemic cardiac disease is the leading cause of death in the developed world. The inability of the adult mammalian heart to adequately repair itself has motivated stem cell researchers to explore various strategies to regenerate cardiomyocytes after myocardial infarction. Over the past century, progressive gains in our knowledge about the cellular mechanisms governing fate determination have led to recent advances in cellular reprogramming. The identification of specific factors capable of inducing pluripotent phenotype in somatic cells as well as factors that can directly reprogram somatic cells into cardiomyocytes suggests the potential for these approaches to translate into clinical therapies in the future. While conceptually appealing, the field of cell lineage reprogramming is in its infancy and further research will be needed to improve the efficiency of the reprogramming process and the fidelity of the reprogrammed cells to their in vivo counterpart. PMID:24764131

  19. RBE for late somatic effects in mice irradiated with 60 MeV protons relative to X-rays.

    NASA Technical Reports Server (NTRS)

    Darden, E. B., Jr.; Clapp, N. K.; Bender, R. S.; Jernigan, M. C.; Upton, A. C.

    1971-01-01

    Investigation of the relative biological effectiveness of energetic protons for the induction of somatic effects in a mammal (mice) following whole body irradiation. The proton energy used approximates the mean energy for proton spectra accompanying solar events. The effects on longevity and the incidence of major neoplastic diseases are summarized. The results obtained suggest that medium energy proton irradiation is no more effective, and on the whole, probably less effective, than conventional X radiation for the induction of late radiation effects in the mouse.

  20. Induction machine

    DOEpatents

    Owen, Whitney H.

    1980-01-01

    A polyphase rotary induction machine for use as a motor or generator utilizing a single rotor assembly having two series connected sets of rotor windings, a first stator winding disposed around the first rotor winding and means for controlling the current induced in one set of the rotor windings compared to the current induced in the other set of the rotor windings. The rotor windings may be wound rotor windings or squirrel cage windings.

  1. Somatic Cell Nuclear Transfer in the Mouse

    NASA Astrophysics Data System (ADS)

    Kishigami, Satoshi; Wakayama, Teruhiko

    Somatic cell nuclear transfer (SCNT) has become a unique and powerful tool for epigenetic reprogramming research and gene manipulation in animals since “Dolly,” the first animal cloned from an adult cell was reported in 1997. Although the success rates of somatic cloning have been inefficient and the mechanism of reprogramming is still largely unknown, this technique has been proven to work in more than 10 mammalian species. Among them, the mouse provides the best model for both basic and applied research of somatic cloning because of its abounding genetic resources, rapid sexual maturity and propagation, minimal requirements for housing, etc. This chapter describes a basic protocol for mouse cloning using cumulus cells, the most popular cell type for NT, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. In particular, we focus on a new, more efficient mouse cloning protocol using trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, which increases both in vitro and in vivo developmental rates from twofold to fivefold. This new method including TSA will be helpful to establish mouse cloning in many laboratories.

  2. Attentional modulation of visceral and somatic pain.

    PubMed

    Dunckley, P; Aziz, Q; Wise, R G; Brooks, J; Tracey, I; Chang, L

    2007-07-01

    A better understanding of the cortical processes underlying attentional modulation of visceral and somatic pain in health are essential for interpretation of future imaging studies of hypervigilance towards bodily sensations which is considered to be an aetiologically important factor in the heightened pain reported by patients with irritable bowel syndrome and fibromyalgia. Twelve healthy subjects were recruited for this study. Simultaneous trains of electrical pulses (delivered to either the rectum or lower abdomen) and auditory tones lasting 6 s were delivered to the subjects during a whole-brain functional scan acquisition. Subjects were instructed to attend to the auditory tones (distracter task) or electrical pulses (pain task). Pain intensity ratings were significantly lower during the distraction task compared with the pain task (P < 0.01) in both sensory modalities. The left primary somatosensory cortex increased in activity with increasing pain report, during attention to visceral pain. Bilateral anterior insula (aIns) cortex activity increased with increasing somatic pain report independent of the direction of attention. Conversely, the primary and secondary auditory cortices significantly increased in activation with decreased pain report. These results suggest that pain intensity perception during attentional modulation is reflected in the primary somatosensory cortex (visceral pain) and aIns cortex activity (somatic pain). PMID:17593138

  3. By-products of immunoglobulin somatic hypermutation.

    PubMed

    Bemark, Mats; Neuberger, Michael S

    2003-09-01

    The antigen receptor loci are the only loci in humans to undergo programmed somatic gene modification. Although aberrant V(D)J integration and class switch recombination can both give rise to chromosomal translocations, a role for somatic hypermutation in such genomic rearrangements has been suggested but is less clearly established. To characterize the types of by-products of somatic hypermutation, we analyzed aberrant rearrangements involving the immunoglobulin loci in a human B-cell line (Ramos) that performs Ig V gene hypermutation constitutively during culture. Single-nucleotide substitutions account for 95% of the mutational events in the VH gene, with small deletions and duplications accounting for most of the remaining mutations. However, larger genetic alterations can be detected at low frequency, accounting for 0.5% of VH-inactivating mutations. Examples include a large (13 kb) deletion, which entirely removes the expressed VH gene; a 3-kb deletion extending from the functional VHDJH into an upstream VH (generating a hybrid VHDJH gene reminiscent of VH replacement); and an ectopic insertion into the VH from chromosome 1. The results support the proposal that aberrant antibody hypermutation can lead to gross genomic alterations but indicate that such events are rare. PMID:12874784

  4. Effect of ovary induction on bread wheat anther culture: ovary genotype and developmental stage, and candidate gene association

    PubMed Central

    Castillo, Ana M.; Sánchez-Díaz, Rosa A.; Vallés, María P.

    2015-01-01

    Ovary pre-conditioned medium and ovary co-culture increased the efficiency of green doubled haploid plant production in bread wheat anther culture. The positive effect of this medium led to a 6- and 11-fold increase in the numbers of embryos and green plants, respectively, having a greater effect on a medium-low responding cultivar. Ovary genotype and developmental stage significantly affected microspore embryogenesis. By the use of Caramba ovaries it was possible to reach a 2-fold increase in the number of embryos and green plants, and to decrease the rate of albinism. Mature ovaries from flowers containing microspores at a late binucleate stage raised the number of embryos and green plants by 25–46% as compared to immature ovaries (excised from flowers with microspores at a mid-late uninucleate stage). The highest numbers of embryos and green plants were produced when using mature Caramba ovaries. Ovaries from Galeón, Tigre, and Kilopondio cultivars successfully induced microspore embryogenesis at the same rate as Caramba ovaries. Moreover, Tigre ovaries raised the percentage of spontaneous chromosome doubling up to 71%. Attempts were made to identify molecular mechanisms associated to the inductive effect of the ovaries on microspore embryogenesis. The genes TAA1b, FLA26, and WALI6 associated to wheat microspore embryogenesis, the CGL1 gene involved in glycan biosynthesis or degradation, and the FER gene involved in the ovary signaling process were expressed and/or induced at different rates during ovary culture. The expression pattern of FLA26 and FER could be related to the differences between genotypes and developmental stages in the inductive effect of the ovary. Our results open opportunities for new approaches to increase bread wheat doubled haploid production by anther culture, and to identify the functional components of the ovary inductive effect on microspore embryogenesis. PMID:26150821

  5. Effect of ovary induction on bread wheat anther culture: ovary genotype and developmental stage, and candidate gene association.

    PubMed

    Castillo, Ana M; Sánchez-Díaz, Rosa A; Vallés, María P

    2015-01-01

    Ovary pre-conditioned medium and ovary co-culture increased the efficiency of green doubled haploid plant production in bread wheat anther culture. The positive effect of this medium led to a 6- and 11-fold increase in the numbers of embryos and green plants, respectively, having a greater effect on a medium-low responding cultivar. Ovary genotype and developmental stage significantly affected microspore embryogenesis. By the use of Caramba ovaries it was possible to reach a 2-fold increase in the number of embryos and green plants, and to decrease the rate of albinism. Mature ovaries from flowers containing microspores at a late binucleate stage raised the number of embryos and green plants by 25-46% as compared to immature ovaries (excised from flowers with microspores at a mid-late uninucleate stage). The highest numbers of embryos and green plants were produced when using mature Caramba ovaries. Ovaries from Galeón, Tigre, and Kilopondio cultivars successfully induced microspore embryogenesis at the same rate as Caramba ovaries. Moreover, Tigre ovaries raised the percentage of spontaneous chromosome doubling up to 71%. Attempts were made to identify molecular mechanisms associated to the inductive effect of the ovaries on microspore embryogenesis. The genes TAA1b, FLA26, and WALI6 associated to wheat microspore embryogenesis, the CGL1 gene involved in glycan biosynthesis or degradation, and the FER gene involved in the ovary signaling process were expressed and/or induced at different rates during ovary culture. The expression pattern of FLA26 and FER could be related to the differences between genotypes and developmental stages in the inductive effect of the ovary. Our results open opportunities for new approaches to increase bread wheat doubled haploid production by anther culture, and to identify the functional components of the ovary inductive effect on microspore embryogenesis. PMID:26150821

  6. Correction: Somatic mutations in cancer development

    PubMed Central

    2011-01-01

    Since publication of Environmental Health 2011, 10(Suppl 1):S12 [1] it has been noticed that titles and captions for the figures and tables were incorrectly applied. In this full-length correction article, figures and tables have been renumbered with legends and captions applied appropriately. Some minor typographical errors have also been corrected. The inconvenience caused to readers by premature publication of the original paper is regretted. The transformation of a normal cell into a cancer cell takes place through a sequence of a small number of discrete genetic events, somatic mutations: thus, cancer can be regarded properly as a genetic disease of somatic cells. The analogy between evolution of organisms and evolution of cell populations is compelling: in both cases what drives change is mutation, but it is Darwinian selection that enables clones that have a growth advantage to expand, thus providing a larger target size for the next mutation to hit. The search for molecular lesions in tumors has taken on a new dimension thanks to two powerful technologies: the micro-arrays for quantitative analysis of global gene expresssion (the transcriptome); and ‘deep’ sequencing for the global analysis of the entire genome (or at least the exome). The former offers the most complete phenotypic characterization of a tumor we could ever hope for – we could call this the ultimate phenotype; the latter can identify all the somatic mutations in an individual tumor – we could call this the somatic genotype. However, there is definitely the risk that while we are ‘drowned by data, we remain thirsty for knowledge’. If we want to heed the teachings of Lorenzo Tomatis, I think the message is clear: we ought to take advantage of the new powerful technologies – not by becoming their slaves, but remaining their masters. Identifying somatic mutations in a tumor is important because through a deeper understanding of the nature of that particular tumor it can help us to optimize therapy or to design new therapeutic approaches.

  7. Shoot regeneration and embryogenesis in lily shoot tips cryopreserved by droplet vitrification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shoot regeneration and embryogenesis were, for the first time, achieved directly in shoot tips of Lilium Oriental hybrid ‘Siberia’ following cryopreservation by droplet-vitrification. Shoot tips (2 mm in length) including 2-3 leaf primordia were excised from 4-week-old adventitious shoots directly r...

  8. The Pesticide Malathion Disrupts "Xenopus" and Zebrafish Embryogenesis: An Investigative Laboratory Exercise in Developmental Toxicology

    ERIC Educational Resources Information Center

    Chemotti, Diana C.; Davis, Sarah N.; Cook, Leslie W.; Willoughby, Ian R.; Paradise, Christopher J.; Lom, Barbara

    2006-01-01

    Malathion is an organophosphorus insecticide, which is often sprayed to control mosquitoes. When applied to aquatic habitats, malathion can also influence the embryogenesis of non-target organisms such as frogs and fish. We modified the frog embryo teratogen assay in "Xenopus" (FETAX), a standard toxicological assay, into an investigative…

  9. Germ-granule components prevent somatic development in the C. elegans germline

    PubMed Central

    Knutson, Andrew Kekūpa'a; Egelhofer, Thea A.; Campbell, Anne C.; Strome, Susan

    2014-01-01

    Summary Specialized ribonucleoprotein organelles collectively known as germ granules are found in the germline cytoplasm from worms to humans [1]. In Drosophila, germ granules have been implicated in germline determination [2]. C. elegans germ granules, known as P granules, do not appear to be required for primordial germ cell (PGC) determination [3], but their components are still needed for fertility [4–6]. One potential role for P granules is to maintain germline fate and totipotency. This is suggested by the loss of P granules from germ cells that transform into somatic cell types, e.g. in germlines lacking MEX-3 and GLD-1 or upon neuronal induction by CHE-1 [7, 8]. However, it has not been established whether loss of P granules is the cause or effect of cell-fate transformation. To test cause-effect, we severely compromised P granules by simultaneously knocking down factors that nucleate granule formation (PGL-1 and PGL-3) and promote their perinuclear localization (GLH-1 and GLH-4) [9], and investigated if that causes germ cells to lose totipotency and initiate somatic reprogramming. We found that compromising P granules causes germ cells to express neuronal and muscle markers and send out neurite-like projections, suggesting that P granules maintain totipotency and germline identity by antagonizing somatic fate. PMID:24746798

  10. In-depth Proteomics Characterization of Embryogenesis of the Honey Bee Worker (Apis mellifera ligustica) *

    PubMed Central

    Fang, Yu; Feng, Mao; Han, Bin; Lu, Xiaoshan; Ramadan, Haitham; Li, Jianke

    2014-01-01

    Identifying proteome changes of honey bee embryogenesis is of prime importance for unraveling the molecular mechanisms that they underlie. However, many proteomic changes during the embryonic period are not well characterized. We analyzed the proteomic alterations over the complete time course of honey bee worker embryogenesis at 24, 48, and 72 h of age, using mass spectrometry-based proteomics, label-free quantitation, and bioinformatics. Of the 1460 proteins identified the embryo of all three ages, the core proteome (proteins shared by the embryos of all three ages, accounting for 40%) was mainly involved in protein synthesis, metabolic energy, development, and molecular transporter, which indicates their centrality in driving embryogenesis. However, embryos at different developmental stages have their own specific proteome and pathway signatures to coordinate and modulate developmental events. The young embryos (<24 h) stronger expression of proteins related to nutrition storage and nucleic acid metabolism may correlate with the cell proliferation occurring at this stage. The middle aged embryos (24–48 h) enhanced expression of proteins associated with cell cycle control, transporters, antioxidant activity, and the cytoskeleton suggest their roles to support rudimentary organogenesis. Among these proteins, the biological pathways of aminoacyl-tRNA biosynthesis, β-alanine metabolism, and protein export are intensively activated in the embryos of middle age. The old embryos (48–72 h) elevated expression of proteins implicated in fatty acid metabolism and morphogenesis indicate their functionality for the formation and development of organs and dorsal closure, in which the biological pathways of fatty acid metabolism and RNA transport are highly activated. These findings add novel understanding to the molecular details of honey bee embryogenesis, in which the programmed activation of the proteome matches with the physiological transition observed during embryogenesis. The identified biological pathways and key node proteins allow for further functional analysis and genetic manipulation for both the honey bee embryos and other eusocial insects. PMID:24895377

  11. Contrasting Storage Protein Synthesis and Messenger RNA Accumulation during Development of Zygotic and Somatic Embryos of Alfalfa (Medicago sativa L.) 1

    PubMed Central

    Krochko, Joan E.; Pramanik, Saroj K.; Bewley, J. Derek

    1992-01-01

    During development on hormone-free media, somatic embryos pass through distinct morphological stages that superficially resemble those of zygotic embryo development (globular, heart, torpedo, cotyledonary stages). Despite these similarities, they differ from zygotic embryos in the extent of cotyledonary development and the patterns of synthesis and quantitative expression of seed-specific storage proteins (7S, 11S, and 2S proteins). Alfin (7S) is the first storage protein synthesized in developing zygotic embryos (stage IV). The 11S (medicagin) and 2S (Low Molecular Weight, LMW) storage proteins are not detectable until the following stage of development (stage V), although all three are present before the completion of embryo enlargement. Likewise, the 7S storage protein is the first to be synthesized in developing somatic embryos (day 5). Medicagin is evident by day 7 and the LMW protein by day 10. In contrast to zygotic embryos, alfin remains the predominant storage protein in somatic embryos throughout development. Not only are the relative amounts of medicagin and the LMW protein reduced in somatic embryos but the LMW protein is accumulated much later than the other proteins. Quantification of the storage protein mRNAs (7S, 11S, and 2S) by northern blot analysis confirms that there are substantial differences in the patterns of message accumulation in zygotic and somatic embryos of alfalfa (Medicago sativa). In zygotic embryos, the 7S, 11S, and 2S storage protein mRNAs are abundant during maturation and, in particular, during the stages of maximum protein synthesis (alfin, stages VI and VII; medicagin, stage VII; LMW, stage VII). In somatic embryos, the predominance of the 7S storage protein is correlated with increased accumulation of its mRNA, whereas the limited synthesis of the 11S storage protein is associated with much lower steady-state levels of its message. The mRNA for the LMW protein is present already by 3 days after transfer to hormone-free media, yet that protein is not evident on stained gels until day 10. Thus, both transcriptional and posttranscriptional events appear to be important in determining the protein complement of these seed tissues. On the basis of storage protein and mRNA accumulation, mature (14 days) somatic embryos most closely resemble stage VI zygotic embryos. The results of the developmental comparison also suggest that the patterns of synthesis of the individual storage proteins (7S, 11S, or 2S) are regulated independently of each other during embryogenesis in alfalfa. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5Figure 6 PMID:16668882

  12. Human somatic mutation assays as biomarkers of carcinogenesis.

    PubMed Central

    Compton, P J; Hooper, K; Smith, M T

    1991-01-01

    This paper describes four assays that detect somatic gene mutations in humans: the hypoxanthine-guanine phosphoribosyl transferase assay, the glycophorin A assay, the HLA-A assay, and the sickle cell hemoglobin assay. Somatic gene mutation can be considered a biomarker of carcinogenesis, and assays for somatic mutation may assist epidemiologists in studies that attempt to identify factors associated with increased risks of cancer. Practical aspects of the use of these assays are discussed. PMID:1954924

  13. Human somatic mutation assays as biomarkers of carcinogenesis

    SciTech Connect

    Compton, P.J.E.; Smith, M.T. ); Hooper, K. )

    1991-08-01

    This paper describes four assays that detect somatic gene mutations in humans: the hypoxanthine-guanine phosphoribosyl transferase assay, the glycophorin A assay, the HLA-A assay, and the sickle cell hemoglobin assay. Somatic gene mutations can be considered a biomarker of carcinogenesis, and assays for somatic mutation may assist epidemiologists in studies that attempt to identify factors associated with increased risks of cancer. Practical aspects of the use of these assays are discussed.

  14. Ancient origin of somatic and visceral neurons

    PubMed Central

    2013-01-01

    Background A key to understanding the evolution of the nervous system on a large phylogenetic scale is the identification of homologous neuronal types. Here, we focus this search on the sensory and motor neurons of bilaterians, exploiting their well-defined molecular signatures in vertebrates. Sensorimotor circuits in vertebrates are of two types: somatic (that sense the environment and respond by shaping bodily motions) and visceral (that sense the interior milieu and respond by regulating vital functions). These circuits differ by a small set of largely dedicated transcriptional determinants: Brn3 is expressed in many somatic sensory neurons, first and second order (among which mechanoreceptors are uniquely marked by the Brn3+/Islet1+/Drgx+ signature), somatic motoneurons uniquely co-express Lhx3/4 and Mnx1, while the vast majority of neurons, sensory and motor, involved in respiration, blood circulation or digestion are molecularly defined by their expression and dependence on the pan-visceral determinant Phox2b. Results We explore the status of the sensorimotor transcriptional code of vertebrates in mollusks, a lophotrochozoa clade that provides a rich repertoire of physiologically identified neurons. In the gastropods Lymnaea stagnalis and Aplysia californica, we show that homologues of Brn3, Drgx, Islet1, Mnx1, Lhx3/4 and Phox2b differentially mark neurons with mechanoreceptive, locomotory and cardiorespiratory functions. Moreover, in the cephalopod Sepia officinalis, we show that Phox2 marks the stellate ganglion (in line with the respiratory — that is, visceral— ancestral role of the mantle, its target organ), while the anterior pedal ganglion, which controls the prehensile and locomotory arms, expresses Mnx. Conclusions Despite considerable divergence in overall neural architecture, a molecular underpinning for the functional allocation of neurons to interactions with the environment or to homeostasis was inherited from the urbilaterian ancestor by contemporary protostomes and deuterostomes. PMID:23631531

  15. Mechanisms and models of somatic cell reprogramming

    PubMed Central

    Buganim, Yosef; Faddah, Dina A.; Jaenisch, Rudolf

    2014-01-01

    Conversion of somatic cells to pluripotency by defined factors is a long and complex process that yields embryonic stem cell-like cells that vary in their developmental potential. To improve the quality of resulting induced pluripotent stem cells (iPSCs), which is important for potential therapeutic applications, and to address fundamental questions about control of cell identity, molecular mechanisms of the reprogramming process must be understood. Here we discuss recent discoveries regarding the role of reprogramming factors in remodeling the genome, including new insights into the function of c-Myc, and describe the different phases, markers and emerging models of reprogramming. PMID:23681063

  16. [Stress theories and the somatization process].

    PubMed

    Dantzer, R

    1995-12-01

    Stress theories aim at understanding pathophysiology of psychosomatic disorders. The first stress theories have been inspired by the principles of homeostasis. They view the response to stressors as a quasi reflex reaction which aims at normalizing disturbed homeostasis. More modern stress theories emphasize the intermediate role of cognitive and behavioural processes in the determinism of neuroendocrine and neurovegetative responses to stressors. Active attempts to control the situation are associated with activation of the sympathetic and adrenal medullary system whereas loss of control is associated with activation of the hypothalamic-pituitary-adrenal axis. Since the functional consequences of the activation of each of these physiological systems are not the same, the risk factors corresponding to each coping strategy are not the same. Whatever their details, physiological and psychobiological stress theories all emphasize the influence of psychic factors on bodily functions. However, mental states do not function independently of bodily functions. In the case of the influences of stress on immunity for instance, it has been shown that these influences represent the counterpart of feedback regulatory mechanisms in which the ability of the brain to regulate immune responses depends on the capacity of the immune system to influence brain functions. Activation of the immune system during infection or inflammation is accompanied by profound metabolic, neuroendocrine and behavioural changes which are mediated by the effects of immune products known as cytokines on brain cell targets. In view of the reciprocal relationships between peripheral organic systems and the brain, a purely psychosomatic view, from the psyche to the soma, is therefore no longer tenable. In addition, biological accounts of somatization processes run into the risk of minimizing the importance of perception and representation of somatic symptoms. Amplification of somatic symptoms is a common feature of neuroticism or negative affectivity and it bears no relationship with objective pathology. In order to understand somatization processes, there is therefore a need to study how the brain processes information it receives from the body and the way this information competes with information from the external environment. PMID:8929672

  17. Repression of somatic cell fate in the germline.

    PubMed

    Robert, Valérie J; Garvis, Steve; Palladino, Francesca

    2015-10-01

    Germ cells must transmit genetic information across generations, and produce gametes while also maintaining the potential to form all cell types after fertilization. Preventing the activation of somatic programs is, therefore, crucial to the maintenance of germ cell identity. Studies in Caenorhabditis elegans, Drosophila melanogaster, and mouse have revealed both similarities and differences in how somatic gene expression is repressed in germ cells, thereby preventing their conversion into somatic tissues. This review will focus on recent developments in our understanding of how global or gene-specific transcriptional repression, chromatin regulation, and translational repression operate in the germline to maintain germ cell identity and repress somatic differentiation programs. PMID:26043973

  18. Somatization in survivors of catastrophic trauma: a methodological review.

    PubMed Central

    North, Carol S

    2002-01-01

    The literature on mental health effects of catastrophic trauma such as community disasters focuses on posttraumatic stress disorder. Somatization disorder is not listed among the classic responses to disaster, nor have other somatoform disorders been described in this literature. Nondiagnostic "somatization," "somatization symptoms," and "somatic symptoms" form the basis of most information about somatization in the literature. However, these concepts have not been validated, and therefore this work suffers from multiple methodological problems of ascertainment and interpretation. Future research is encouraged to consider many methodological issues in obtaining adequate data to address questions about the association of somatization with traumatic events, including a) appropriate comparison groups, b) satisfactory definition and measurement of somatization, c) exclusion of medical explanations for the symptoms, d) recognition of somatizers' spurious attribution of symptoms to medical causes, e) collection of data from additional sources beyond single-subject interviews, f) validation of diagnosis-unrelated symptom reporting or reconsideration of symptoms within diagnostic frameworks, g) separation of somatization after an event into new (incident) and preexisting categories, h) development of research models that include sufficient variables to examine the broader scope of potential relationships, and i) novel consideration of alternative causal directionalities. PMID:12194899

  19. Clarifying tetrapod embryogenesis, a physicist's point of view

    NASA Astrophysics Data System (ADS)

    Fleury, V.

    2009-03-01

    The origin of tetrapods is a complex question that webs together genetic, paleontological, developmental and physical facts. Basically, the development of embryos is described by a complex mix of mechanical movements and biochemical inductions of genetic origin. It is difficult to sort out in this scientific question what are the fundamental features imposed by conservation laws of physics, and by force equilibria, and what can be ascribed to successive, very specific, stop-and-go inductions of genetic nature. A posteriori, evolution selects the parameters of this process as found in the observed species. Whether there is a general law to animal formation seems out of the question. However, several concepts developed in biology, like the concept of “organizer” seem questionable from a physics point of view, since the entire deformation and force field should be the “organizer” of development, and one can hardly ascribe such a role to a single small area of the embryo body. In the same spirit, the concept of “positional information” encapsulated in concentration of chemicals seems questionable since the deformation and force fields in embryonic tissues are tensors. Finally, the concept of a development organized in space along three orthogonal (“Cartesian”) axes associated to chemical gradients seems also questionable, since early embryo development is driven by complex vortex fields, with hyperbolic trajectories which span the entire embryo. Such hyperbolic trajectories are best understood by a description in terms of dipolar components of the morphogenetic forces, whose projections along orthogonal axe have no specific meaning except as a mathematical tool. I review here the present state of description of several aspects of tetrapods morphogenesis and evolution, from the point of view of physics. It is getting clear that several basic features of tetrapods body are a direct consequences of fundamental laws of physics. Several lines of work reviewed here show that the topology of the tetrapods may be directly related to the structure of the earliest movements in embryos. The bio-mechanical approach leads to important consequences for the constraints on evolution of the craniates. Such consequences have received a controversial welcome in the last decade, although they may encapsulate the true origin of craniates, esp. simians, and eventually homo.

  20. Evaluation of Nine Somatic Variant Callers for Detection of Somatic Mutations in Exome and Targeted Deep Sequencing Data.

    PubMed

    Krøigård, Anne Bruun; Thomassen, Mads; Lænkholm, Anne-Vibeke; Kruse, Torben A; Larsen, Martin Jakob

    2016-01-01

    Next generation sequencing is extensively applied to catalogue somatic mutations in cancer, in research settings and increasingly in clinical settings for molecular diagnostics, guiding therapy decisions. Somatic variant callers perform paired comparisons of sequencing data from cancer tissue and matched normal tissue in order to detect somatic mutations. The advent of many new somatic variant callers creates a need for comparison and validation of the tools, as no de facto standard for detection of somatic mutations exists and only limited comparisons have been reported. We have performed a comprehensive evaluation using exome sequencing and targeted deep sequencing data of paired tumor-normal samples from five breast cancer patients to evaluate the performance of nine publicly available somatic variant callers: EBCall, Mutect, Seurat, Shimmer, Indelocator, Somatic Sniper, Strelka, VarScan 2 and Virmid for the detection of single nucleotide mutations and small deletions and insertions. We report a large variation in the number of calls from the nine somatic variant callers on the same sequencing data and highly variable agreement. Sequencing depth had markedly diverse impact on individual callers, as for some callers, increased sequencing depth highly improved sensitivity. For SNV calling, we report EBCall, Mutect, Virmid and Strelka to be the most reliable somatic variant callers for both exome sequencing and targeted deep sequencing. For indel calling, EBCall is superior due to high sensitivity and robustness to changes in sequencing depths. PMID:27002637

  1. Evaluation of Nine Somatic Variant Callers for Detection of Somatic Mutations in Exome and Targeted Deep Sequencing Data

    PubMed Central

    Krøigård, Anne Bruun; Thomassen, Mads; Lænkholm, Anne-Vibeke; Kruse, Torben A.; Larsen, Martin Jakob

    2016-01-01

    Next generation sequencing is extensively applied to catalogue somatic mutations in cancer, in research settings and increasingly in clinical settings for molecular diagnostics, guiding therapy decisions. Somatic variant callers perform paired comparisons of sequencing data from cancer tissue and matched normal tissue in order to detect somatic mutations. The advent of many new somatic variant callers creates a need for comparison and validation of the tools, as no de facto standard for detection of somatic mutations exists and only limited comparisons have been reported. We have performed a comprehensive evaluation using exome sequencing and targeted deep sequencing data of paired tumor-normal samples from five breast cancer patients to evaluate the performance of nine publicly available somatic variant callers: EBCall, Mutect, Seurat, Shimmer, Indelocator, Somatic Sniper, Strelka, VarScan 2 and Virmid for the detection of single nucleotide mutations and small deletions and insertions. We report a large variation in the number of calls from the nine somatic variant callers on the same sequencing data and highly variable agreement. Sequencing depth had markedly diverse impact on individual callers, as for some callers, increased sequencing depth highly improved sensitivity. For SNV calling, we report EBCall, Mutect, Virmid and Strelka to be the most reliable somatic variant callers for both exome sequencing and targeted deep sequencing. For indel calling, EBCall is superior due to high sensitivity and robustness to changes in sequencing depths. PMID:27002637

  2. Susceptible periods during embryogenesis of the heart and endocrine glands.

    PubMed Central

    Sadler, T W

    2000-01-01

    One of the original principles of teratology states that, "Susceptibility to teratogenesis varies with the developmental stage at the time of exposure to an adverse influence" [Wilson JG. Environment and Birth Defects. New York:Academic Press, 1973]. The time of greatest sensitivity encompasses the period of organ formation during weeks 3-8 following fertilization in human gestation. At this time, stem cell populations for each organ's morphogenesis are established and inductive events for the initiation of differentiation occur. Structural defects of the heart and endocrine system are no exception to this axiom and have their origins during this time frame. Although the function and maturation of these organs may be affected at later stages, structural defects and loss of cell types usually occur during these early phases of development. Thus, to determine critical windows for studying mechanisms of teratogenesis, it is essential to understand the developmental processes that establish these organs. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 PMID:10852854

  3. Light-emitting diodes and their potential in callus growth, plantlet development and saponin accumulation during somatic embryogenesis of Panax vietnamensis Ha et Grushv.

    PubMed Central

    Nhut, Duong Tan; Huy, Nguyen Phuc; Tai, Ngo Thanh; Nam, Nguyen Ba; Luan, Vu Quoc; Hien, Vu Thi; Tung, Hoang Thanh; Vinh, Bui The; Luan, Tran Cong

    2015-01-01

    In recent years, LED (light-emitting diode) has been the subject of research within the field of plant growth and development. However, there has been little discussion about using LED in vitro cultures of Panax vietnamensis, one of the important medicinal plants belonging to the Panax genus. This study examines the influence of various LED lamps on callus growth and plant formation of P. vietnamensis. Results show significant differences in growth and development, as various light conditions were suitable for different stages. Callus of 70 mg in fresh weight cultured under yellow LEDs resulted in growth of 1197 mg in fresh weight and 91.7 mg of dry weight, within a period of three months. The most effective plant formation was obtained when embryogenic calli were cultured under the combination of 60% red LED and 40% blue LED with an average of 11.21 plantlets per explant; the shoot clump fresh weight and dry weight were of 1147 and 127 mg, respectively, and the average plant height was 3.1 cm. It was also shown that this light condition was the most efficient for P. vietnamensis in vitro plant growth and development. This study provided additional evidence regarding the influence of different LEDs on ginsenoside production applying high-performance liquid chromatography (HPLC) analysis with photo-diode array (PDA) detection at ultraviolet (UV) wavelength 203 nm. The highest MR2 content was recorded when plants maintained under 20% red LED combined with 80% blue LED. However, the highest Rg1 and Rb1 content was found under fluorescent light. The results presented might provide new strategies using LEDs for adequate micropropagation protocols of P. vietnamensis. PMID:26019644

  4. The IMD innate immunity pathway of Drosophila influences somatic sex determination via regulation of the Doa locus.

    PubMed

    Zhao, Yunpo; Cocco, Claudia; Domenichini, Severine; Samson, Marie-Laure; Rabinow, Leonard

    2015-11-15

    The IMD pathway induces the innate immune response to infection by gram-negative bacteria. We demonstrate strong female-to-male sex transformations in double mutants of the IMD pathway in combination with Doa alleles. Doa encodes a protein kinase playing a central role in somatic sex determination through its regulation of alternative splicing of dsx transcripts. Transcripts encoding two specific Doa isoforms are reduced in Rel null mutant females, supporting our genetic observations. A role for the IMD pathway in somatic sex determination is further supported by the induction of female-to-male sex transformations by Dredd mutations in sensitized genetic backgrounds. In contrast, mutations in either dorsal or Dif, the two other NF-κB paralogues of Drosophila, display no effects on sex determination, demonstrating the specificity of IMD signaling. Our results reveal a novel role for the innate immune IMD signaling pathway in the regulation of somatic sex determination in addition to its role in response to microbial infection, demonstrating its effects on alternative splicing through induction of a crucial protein kinase. PMID:26434917

  5. Study of model systems to test the potential function of Artemia group 1 late embryogenesis abundant (LEA) proteins.

    PubMed

    Warner, Alden H; Guo, Zhi-hao; Moshi, Sandra; Hudson, John W; Kozarova, Anna

    2016-01-01

    Embryos of the brine shrimp, Artemia franciscana, are genetically programmed to develop either ovoviparously or oviparously depending on environmental conditions. Shortly upon their release from the female, oviparous embryos enter diapause during which time they undergo major metabolic rate depression while simultaneously synthesize proteins that permit them to tolerate a wide range of stressful environmental events including prolonged periods of desiccation, freezing, and anoxia. Among the known stress-related proteins that accumulate in embryos entering diapause are the late embryogenesis abundant (LEA) proteins. This large group of intrinsically disordered proteins has been proposed to act as molecular shields or chaperones of macromolecules which are otherwise intolerant to harsh conditions associated with diapause. In this research, we used two model systems to study the potential function of the group 1 LEA proteins from Artemia. Expression of the Artemia group 1 gene (AfrLEA-1) in Escherichia coli inhibited growth in proportion to the number of 20-mer amino acid motifs expressed. As well, clones of E. coli, transformed with the AfrLEA-1 gene, expressed multiple bands of LEA proteins, either intrinsically or upon induction with isopropyl-β-thiogalactoside (IPTG), in a vector-specific manner. Expression of AfrLEA-1 in E. coli did not overcome the inhibitory effects of high concentrations of NaCl and KCl but modulated growth inhibition resulting from high concentrations of sorbitol in the growth medium. In contrast, expression of the AfrLEA-1 gene in Saccharomyces cerevisiae did not alter the growth kinetics or permit yeast to tolerate high concentrations of NaCl, KCl, or sorbitol. However, expression of AfrLEA-1 in yeast improved its tolerance to drying (desiccation) and freezing. Under our experimental conditions, both E. coli and S. cerevisiae appear to be potentially suitable hosts to study the function of Artemia group 1 LEA proteins under environmentally stressful conditions. PMID:26462928

  6. Release of somatic embryogenic potential from excised zygotic embryos of carrot and maintenance of proembryonic cultures in hormone-free medium

    NASA Technical Reports Server (NTRS)

    Smith, D. L.; Krikorian, A. D.

    1989-01-01

    Excised zygotic embryos, mericarps ("seeds") and hypocotyls of seedlings of cultivated carrot Daucus carota cv. Scarlet Nantes were evaluated for their ability to generate somatic embryos on a semisolid hormone-free nutrient medium. Neither intact zygotic embryos nor hypocotyls ever produced somatic embryos. However, mericarps and broken zygotic embryos were excellent sources for somatic embryo production (response levels as high as 86%). Somatic embryo formation was highest from cotyledons, but was also observed on isolated hypocotyls and root tips of mature zygotic embryos. On media containing unreduced nitrogen, somatic embryo formation led to the generation of vigorous cultures comprised entirely of somatic embryos at various stages of development which in turn proliferated still other somatic embryos. However, a medium was devised which when 1-5 mM NH4+ was the sole nitrogen source, led only to a proliferation of globular proembryos. Sustained subculturing of these proembryos at 2-3 week intervals enabled establishment of highly uniform cultures in which no further development into more mature stages of embryonic development occurred. These have been maintained, without decline, as morphogenetically competent proembryonic globules for over ten months. A basal medium containing from 1-5 mM NH4+ as the sole nitrogen source appears not to be inductive to somatic proembryo formation. Instead, such a medium is best thought of as permissive to the expression of embryogenically determined cells within zygotic embryos. By excising and breaking or wounding zygotic embryos, constituent cells are probably released from positional or chemical restraints and thus are able to express their innate embryogenic potential. Once a proembryonic culture is established, this medium containing 1-5 mM NH4+ as the sole nitrogen source provides a nonpermissive environment to the development and growth of later embryonic stages, but it does allow the continued formation and multiplication of globular somatic proembryos. The sequence of events leading from excised broken zygotic embryos to the formation of somatic embryos and the maintenance of somatic proembryos are demonstrated by scanning electron microscopy and histological preparations. Germination levels from intact zygotic embryos on media with varying levels and ratios of unreduced vs. reduced inorganic nitrogen were determined as well and provided baseline or control data on the type of response obtained from nonwounded material.

  7. Notum is required for neural and head induction via Wnt deacylation, oxidation and inactivation

    PubMed Central

    Zhang, Xinjun; Cheong, Seong-Moon; Amado, Nathalia G.; Reis, Alice H.; MacDonald, Bryan T.; Zebisch, Matthias; Jones, E. Yvonne; Abreu, Jose Garcia; He, Xi

    2015-01-01

    Summary Secreted Wnt morphogens are essential for embryogenesis and homeostasis, and require a lipid/palmitoleoylate modification for receptor binding and activity. Notum is a secreted Wnt antagonist that belongs to the α/β hydrolase superfamily, but its mechanism of action and roles in vertebrate embryogenesis are not fully understood. Here we report that Notum hydrolyzes the Wnt palmitoleoylate adduct extracellularly, resulting in inactivated Wnt proteins that form oxidized oligomers incapable of receptor binding. Thus Notum is a Wnt deacylase, and palmitoleoylation is obligatory for the Wnt structure that maintains its active monomeric conformation. Notum is expressed in naïve ectoderm and neural plate in Xenopus and is required for neural and head induction. These findings suggest that distinct mechanisms of Wnt inactivation by the Tiki protease in the Organizer and the Notum deacylase in presumptive neuroectoderm orchestrate vertebrate brain development. PMID:25771893

  8. Conservation of proteo-lipid nuclear membrane fusion machinery during early embryogenesis

    PubMed Central

    Byrne, Richard D; Veeriah, Selvaraju; Applebee, Christopher J; Larijani, Banafshé

    2014-01-01

    The fusogenic lipid diacylglycerol is essential for remodeling gamete and zygote nuclear envelopes (NE) during early embryogenesis. It is unclear whether upstream signaling molecules are likewise conserved. Here we demonstrate PLCγ and its activator SFK1, which co-operate during male pronuclear envelope formation, also promote the subsequent male and female pronuclear fusion. PLCγ and SFK1 interact directly at the fusion site leading to PLCγ activation. This is accompanied by a spatially restricted reduction of PtdIns(4,5)P2. Consequently, pronuclear fusion is blocked by PLCγ or SFK1 inhibition. These findings identify new regulators of events in the early embryo and suggest a conserved “toolkit” of fusion machinery drives successive NE fusion events during embryogenesis. PMID:25482196

  9. Conservation of proteo-lipid nuclear membrane fusion machinery during early embryogenesis.

    PubMed

    Byrne, Richard D; Veeriah, Selvaraju; Applebee, Christopher J; Larijani, Banafshé

    2014-01-01

    The fusogenic lipid diacylglycerol is essential for remodeling gamete and zygote nuclear envelopes (NE) during early embryogenesis. It is unclear whether upstream signaling molecules are likewise conserved. Here we demonstrate PLCγ and its activator SFK1, which co-operate during male pronuclear envelope formation, also promote the subsequent male and female pronuclear fusion. PLCγ and SFK1 interact directly at the fusion site leading to PLCγ activation. This is accompanied by a spatially restricted reduction of PtdIns(4,5)P2. Consequently, pronuclear fusion is blocked by PLCγ or SFK1 inhibition. These findings identify new regulators of events in the early embryo and suggest a conserved "toolkit" of fusion machinery drives successive NE fusion events during embryogenesis. PMID:25482196

  10. The δ-cyclin expression at early stages of embryogenesis of Brassica rapa L. under clinorotation

    NASA Astrophysics Data System (ADS)

    Artemenko, O. A.; Popova, A. F.

    We present some results of comparison studying of Brassica embryo development and the δ-cyclin genes expression under slow horizontal clinorotation and in the laboratory control. Some backlog of the δ1-cyclin genes expression at early stages of embryogenesis under clinorotation was revealed in comparison with the laboratory control. The similar level of the δ3-cyclin expression at all stages of embryo formation (from one to nine days) in both variants is shown. Some delays in the rate of Brassica rapa embryo development under clinorotation in comparison with the laboratory control can be a result of decrease of a level and some backlog of the δ1-cyclin expression at early stages of embryogenesis.

  11. Somatics in the Dance Studio: Embodying Feminist/Democratic Pedagogy

    ERIC Educational Resources Information Center

    Burnidge, Anne

    2012-01-01

    Since the 1970s, somatics have increasingly become a part of the dance training landscape. Although the psychophysical benefits seem sufficient in themselves to warrant inclusion in dance, this article explores another possible outcome of embracing somatic pedagogical principles, a change that affects not "what" is taught in a dance class, but…

  12. Natural Attributes and Agricultural Implications of Somatic Genome Variation.

    PubMed

    Li, Xiu-Qing

    2016-01-01

    This article proposes the concept of genome network, describes different variations of the somatic genome network, and reviews the agricultural implications of such variations. All genetic materials in a cell constitute the genome network of the cell and can jointly influence the cell's function and fate. The somatic genome of a plant is the genome network of cells in somatic tissues and of nonreproductive cells in pollen and ovules. Somatic genome variation (SGV, approximately equivalent to somagenetic variation) occurs at multiple levels, including stoichiometric, ploidy, and sequence variations. For a multicellular organism, the term "somatic genome variation" covers both the variation in part of the organism and the generation of new genotype individuals through somatic means from a sexually produced original genotype. For unicellular organisms, genome variation in somatic nuclei occurs at the whole organism level because there is only a single cell per individual. Growth, development and evolution of living organisms require both stability and instability of their genomes. Somatic genome variation displays many more attributes than genetic mutation and has strong implications for agriculture. PMID:26636317

  13. Human somatic cell reprogramming: does the egg know best?

    PubMed

    Colman, Alan; Burley, Justine

    2014-11-01

    Somatic cell nuclear transfer offers an alternative approach to the use of exogenous transcription factors for the reprogramming of somatic cells. But is it a better way? Two groups have performed detailed molecular comparisons between human cell lines made by the two methods and report different conclusions. PMID:25517458

  14. Development of the Ghent Multidimensional Somatic Complaints Scale

    ERIC Educational Resources Information Center

    Beirens, Koen; Fontaine, Johnny R. J.

    2010-01-01

    The present study aimed at developing a new scale that operationalizes a hierarchical model of somatic complaints. First, 63 items representing a wide range of symptoms and sensations were compiled from somatic complaints scales and emotion literature. These complaints were rated by Belgian students (n = 307) and Belgian adults (n = 603).

  15. Depression, Health, and Somatic Complaints in Older Adults.

    ERIC Educational Resources Information Center

    Mahurin, Kathleen A.; Gatz, Margaret

    Although depression is considered to be common in the elderly, reliable rates of prevalence are lacking. Studies have shown that age differences on measures of depressive symptomatology can be attributed to higher levels of somatic complaints. In order to examine whether the association between somatic and depressive symptoms varies as a function…

  16. Development of the Ghent Multidimensional Somatic Complaints Scale

    ERIC Educational Resources Information Center

    Beirens, Koen; Fontaine, Johnny R. J.

    2010-01-01

    The present study aimed at developing a new scale that operationalizes a hierarchical model of somatic complaints. First, 63 items representing a wide range of symptoms and sensations were compiled from somatic complaints scales and emotion literature. These complaints were rated by Belgian students (n = 307) and Belgian adults (n = 603).…

  17. Somatics in the Dance Studio: Embodying Feminist/Democratic Pedagogy

    ERIC Educational Resources Information Center

    Burnidge, Anne

    2012-01-01

    Since the 1970s, somatics have increasingly become a part of the dance training landscape. Although the psychophysical benefits seem sufficient in themselves to warrant inclusion in dance, this article explores another possible outcome of embracing somatic pedagogical principles, a change that affects not "what" is taught in a dance class, but

  18. Young microspore-derived maize embryos show two domains with defined features also present in zygotic embryogenesis.

    PubMed

    Testillano, Pilar S; Ramrez, Carmen; Domenech, Jezabel; Coronado, Maria-Jos; Vergne, Phillipe; Matthys-Rochon, Elisabeth; Risueo, Mara C

    2002-12-01

    In this work we report the existence of two domains in early microspore maize proembryos displaying similar features of zygotic embryogenesis. The large or so-called endosperm-like domain exhibits specific features: coenocytic organisation, synchronous mitosis, vacuolated cytoplasm, starch granules, incomplete walls containing callose and differential tubuline organisation. The small or embryo-like domain displays small polygonal uninucleate cells with typical organisation of proliferating cells. The structural organisation and the subcellular localization of specific cytoplasmic and cell wall components (starch, tubuline and callose) in both proembryo domains have been determined by using specific cytochemical and immunocytochemical methods. Four morphological types of proembryos containing both domains have been characterised. Taking into account their relative size, the high asynchrony of the culture and the homologies between structural features of endosperm-like domain and zygotic endosperm development, they could represent different stages in microspore embryogenesis development. The ZmAE1 and ZmAE3 genes expressed in the Embryo Surrounding Region of the endosperm during zygotic embryogenesis (Magnard et al., 2000), were revealed to be expressed in early microspore proembryos by in situ hybridization at light and electron microscopy levels. This data supports the existence of an endosperm-like function during early microspore embryogenesis and provides new insights into the onset of microspore embryogenesis in maize, and its parallelism with zygotic embryogenesis. PMID:12533027

  19. Expression of the Xenopus GTP-binding protein gene Ran during embryogenesis.

    PubMed

    Onuma, Y; Nishihara, R; Takahashi, S; Tanegashima, K; Fukui, A; Asashima, M

    2000-06-01

    The Ran gene family encodes small GTP binding proteins that are associated with a variety of nuclear processes. We isolated a Xenopus Ran cDNA and analyzed the pattern of expression of this gene during embryogenesis. Ran is expressed maternally and later in the CNS, neural crest, mesenchyme, eyes, and otic vesicles. However, expression is not detected in the somites or the notochord. PMID:11180838

  20. Convergent occurrence of the developmental hourglass in plant and animal embryogenesis?

    PubMed Central

    Cridge, Andrew G.; Dearden, Peter K.; Brownfield, Lynette R.

    2016-01-01

    Background The remarkable similarity of animal embryos at particular stages of development led to the proposal of a developmental hourglass. In this model, early events in development are less conserved across species but lead to a highly conserved ‘phylotypic period’. Beyond this stage, the model suggests that development once again becomes less conserved, leading to the diversity of forms. Recent comparative studies of gene expression in animal groups have provided strong support for the hourglass model. How and why might such an hourglass pattern be generated? More importantly, how might early acting events in development evolve while still maintaining a later conserved stage? Scope The discovery that an hourglass pattern may also exist in the embryogenesis of plants provides comparative data that may help us explain this phenomenon. Whether the developmental hourglass occurs in plants, and what this means for our understanding of embryogenesis in plants and animals is discussed. Models by which conserved early-acting genes might change their functional role in the evolution of gene networks, how networks buffer these changes, and how that might constrain, or confer diversity, of the body plan are also discused. Conclusions Evidence of a morphological and molecular hourglass in plant and animal embryogenesis suggests convergent evolution. This convergence is likely due to developmental constraints imposed upon embryogenesis by the need to produce a viable embryo with an established body plan, controlled by the architecture of the underlying gene regulatory networks. As the body plan is largely laid down during the middle phases of embryo development in plants and animals, then it is perhaps not surprising this stage represents the narrow waist of the hourglass where the gene regulatory networks are the oldest and most robust and integrated, limiting species diversity and constraining morphological space. PMID:27013176

  1. Hypnotizability and somatic complaints: a gender-specific phenomenon.

    PubMed

    Younger, Jarred W; Rossetti, Gina C; Borckardt, Jeffrey J; Smith, Arthur R; Tasso, Anthony F; Nash, Michael R

    2007-01-01

    The relationship between hypnotizability and somatic illness was measured in 45 college students. Several weeks after completing the Waterloo-Stanford Group C Scale (WSGC), participants filled out a somatic-complaint checklist and measures of psychopathology. Results indicated a positive correlation between hypnotizability and somatic illness, and the relationship was stronger for female participants. In contrast to the quadratic model proposed by Wickramasekera, the current data demonstrated a linear relationship between hypnotizability and somatic complaint. Further analyses showed that somatic complaints were associated with hallucination and imagery items, corresponding to the perceptual-cognitive factor identified in Woody, Barnier, and McConkey's (2005) factor analysis of the Stanford Hypnotic Susceptibility Scale, Form C. The results call into question some claims that high hypnotizability is an adaptive and healthy trait. PMID:17135060

  2. Recent advancements in cloning by somatic cell nuclear transfer

    PubMed Central

    Ogura, Atsuo; Inoue, Kimiko; Wakayama, Teruhiko

    2013-01-01

    Somatic cell nuclear transfer (SCNT) cloning is the sole reproductive engineering technology that endows the somatic cell genome with totipotency. Since the first report on the birth of a cloned sheep from adult somatic cells in 1997, many technical improvements in SCNT have been made by using different epigenetic approaches, including enhancement of the levels of histone acetylation in the chromatin of the reconstructed embryos. Although it will take a considerable time before we fully understand the nature of genomic programming and totipotency, we may expect that somatic cell cloning technology will soon become broadly applicable to practical purposes, including medicine, pharmaceutical manufacturing and agriculture. Here we review recent progress in somatic cell cloning, with a special emphasis on epigenetic studies using the laboratory mouse as a model. PMID:23166393

  3. Nuclear lamins and peripheral nuclear antigens during fertilization and embryogenesis in mice and sea urchins

    SciTech Connect

    Schatten, G.; Schatten, H.; Simerly, C.; Maul, G.G.; Chaly, N.

    1985-07-01

    Nuclear structural changes during fertilization and embryogenesis in mice and sea urchins are traced using four antibodies. The oocytes from virgin female mice, morulae and blastocytes from mated females, and gametes from the sea urchin Lytechnius variegatis are studied using mouse monoclonal antibodies to nuclear lamin A/C, monoclonal antibody to P1, human autoimmune antibodies to lamin A/C, and to lamin B. The mouse fertilization data reveal no lamins on the oocyte; however, lamins are present on the pronuclei, and chromosomes are found on the oocytes and pronuclei. It is detected that on the sea urchin sperm the lamins are reduced to acrosomal and centriolar fossae and peripheral antigens are around the sperm nucleus. The mouse sperm bind lamin antibodies regionally and do not contain antigens. Lamins and antigens are observed on both pronuclei and chromosomes during sea urchin fertilization. Mouse embryogenesis reveals that lamin A/C is not recognized at morula and blastocyst stages; however, lamin B stains are retained. In sea urchin embryogenesis lamin recognition is lost at the blastrula, gastrula, and plutei stages. It is noted that nuclear lamins lost during spermatogenesis are restored at fertilization and peripheral antigens are associated with the surface of chromosomes during meiosis and mitosis and with the periphery of the pronuclei and nuclei during interphase. 32 references.

  4. Oxygen changes drive non-uniform scaling in Drosophila melanogaster embryogenesis

    PubMed Central

    Kuntz, Steven G.; Eisen, Michael B.

    2015-01-01

    We previously demonstrated that, while changes in temperature produce dramatic shifts in the time elapsed during Drosophila melanogaster embryogenesis, the relative timing of events within embryogenesis does not change. However, it was unclear if this uniform scaling is an intrinsic property of developing embryos, or if it is specific to thermal fluctuations. To investigate this, here we characterize the embryonic response to changes in oxygen concentration, which also impact developmental rate, using time-lapse imaging, and find it fundamentally different from the temperature response. Most notably, changes in oxygen levels drive developmental heterochrony, with the timing of several morphological processes showing distinct scaling behaviors. Gut formation is severely slowed by decreases in oxygen, while head involution and syncytial development are less impacted than the rest of development, and the order of several developmental landmarks is inverted at different oxygen levels. These data reveal that the uniform scaling seen with changes in temperature is not a trivial consequence of adjusting developmental rate. The developmental rate changes produced by changing oxygen concentrations dwarf those induced by temperature, and greatly impact survival. While extreme temperatures increase early embryo mortality, mild hypoxia increases arrest and death during mid-embryogenesis and mild hyperoxia increases survival over normoxia. PMID:26673611

  5. Annual Reproductive Cycle and Unusual Embryogenesis of a Temperate Coral in the Mediterranean Sea

    PubMed Central

    Marchini, Chiara; Airi, Valentina; Fontana, Roberto; Tortorelli, Giada; Rocchi, Marta; Falini, Giuseppe; Levy, Oren; Dubinsky, Zvy; Goffredo, Stefano

    2015-01-01

    The variety of reproductive processes and modes among coral species reflects their extraordinary regeneration ability. Scleractinians are an established example of clonal animals that can exhibit a mixed strategy of sexual and asexual reproduction to maintain their populations. This study provides the first description of the annual reproductive cycle and embryogenesis of the temperate species Caryophyllia inornata. Cytometric analyses were used to define the annual development of germ cells and embryogenesis. The species was gonochoric with three times more male polyps than female. Polyps were sexually mature from 6 to 8 mm length. Not only females, but also sexually inactive individuals (without germ cells) and males were found to brood their embryos. Spermaries required 12 months to reach maturity, while oogenesis seemed to occur more rapidly (5–6 months). Female polyps were found only during spring and summer. Furthermore, the rate of gamete development in both females and males increased significantly from March to May and fertilization was estimated to occur from April to July, when mature germ cells disappeared. Gametogenesis showed a strong seasonal influence, while embryos were found throughout the year in males and in sexually inactive individuals without a defined trend. This unusual embryogenesis suggests the possibility of agamic reproduction, which combined with sexual reproduction results in high fertility. This mechanism is uncommon and only four other scleractinians (Pocillopora damicornis, Tubastraea diaphana, T. coccinea and Oulastrea crispata) have been shown to generate their broods asexually. The precise nature of this process is still unknown. PMID:26513159

  6. Sox2/Oct4: A delicately balanced partnership in pluripotent stem cells and embryogenesis.

    PubMed

    Rizzino, Angie; Wuebben, Erin L

    2016-06-01

    Considerable progress has been made in understanding the roles of Sox2 and Oct4 in embryonic stem cells and mammalian embryogenesis. Specifically, significant progress has been made in answering three questions about the functions of Sox2 and Oct4, which are the focus of this review. 1) Are the first or second cell lineage decisions during embryogenesis controlled by Oct4 and/or Sox2? 2) Do the levels of Oct4 and Sox2 need to be maintained within narrow limits to promote normal development and to sustain the self-renewal of pluripotent stem cells? 3) Do Oct4 and Sox2 work closely together or is the primary role of Sox2 in pluripotent cells to ensure the expression of Oct4? Although significant progress has been made in answering these questions, additional studies are needed to resolve several important remaining issues. Nonetheless, the preponderance of the evidence suggests there is considerable crosstalk between Sox2 and Oct4, and further suggests Sox2 and Oct4 function as molecular rheostats and utilize negative feedback loops to carefully balance their expression and other critical genes during embryogenesis. This article is part of a Special Issue entitled: The Oct transcription factor family, edited by Dr. Dean Tantin. PMID:26992828

  7. Developmental biochemistry of cottonseed embryogenesis and germination. XIII. Regulation of biosynthesis of principal storage proteins

    SciTech Connect

    Dure, L. III; Galau, G.A.

    1981-07-01

    The synthesis of the principal cottonseed storage proteins during embryogenesis was followed by analyses of protein synthesized in vivo and from purified RNA in vitro in the wheat germ system. The kinetics of in vivo labeling as well as immunochemical cross-reactivity indicate that the 52- and 48-kilodalton mature storage protein sets are derived from 70- and 67-kilodalton precursor protein sets that are abundant proteins in embryonic cotyledons and disappear in late embryogenesis. Identification of the initial translation products of the storage protein mRNA has not been clearly established although products of apparent molecular weights of 69,000 and 60,000 are the likely storage protein precursors. Storage protein synthesis falls off markedly in late embryogenesis simultaneously with the loss of a superabundant class of mRNAs that are presumed to be those for the storage proteins. The synthesis of these proteins ceases abruptly when immature embryos are removed from the boll and allowed to germinate precociously or when this precocious germination is prevented by incubation in abscisic acid. Thus, abscisic acid is not implicated in the expression of the storage protein genes. A scheme involving co-translational processing into vesicles, glycosylation, and slow in situ cleavage to produce the mature storage proteins is proposed.

  8. Functional analysis of Bombyx Wnt1 during embryogenesis using the CRISPR/Cas9 system.

    PubMed

    Zhang, Zhongjie; Aslam, Abu F M; Liu, Xiaojing; Li, Muwang; Huang, Yongping; Tan, Anjiang

    2015-08-01

    Recently established, custom-designed nuclease technologies such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system provide attractive genome editing tools. Targeted gene mutagenesis using the CRISPR/Cas9 system has been achieved in several orders of insects. However, outside of studies on Drosophila melanogaster and the lepidopteron model insect Bombyx mori, little success has been reported, which is largely due to a lack of effective genetic manipulation tools that can be used in other insect orders. To create a simple and effective method of gene knockout analysis, especially for dissecting gene functioning during insect embryogenesis, we performed a functional analysis of the Bombyx Wnt1 (BmWnt1) gene using Cas9/sgRNA-mediated gene mutagenesis. The Wnt1 gene is required for embryonic patterning in various organisms, and its crucial roles during embryogenesis have been demonstrated in several insect orders. Direct injection of Cas9 mRNA and BmWnt1-specific sgRNA into Bombyx embryos induced a typical Wnt-deficient phenotype: injected embryos could not hatch and exhibited severe defects in body segmentation and pigmentation in a dose-dependent manner. Quantitative real-time PCR (qRT-PCR) analysis revealed that Hox genes were down-regulated after BmWnt1 depletion. Furthermore, large deletion, up to 18Kb, ware generated. The current study demonstrates that using the CRISPR/Cas9 system is a promising approach to achieve targeted gene mutagenesis during insect embryogenesis. PMID:26070541

  9. Evidence for Active Maintenance of Phylotranscriptomic Hourglass Patterns in Animal and Plant Embryogenesis

    PubMed Central

    Drost, Hajk-Georg; Gabel, Alexander; Grosse, Ivo; Quint, Marcel

    2015-01-01

    The developmental hourglass model has been used to describe the morphological transitions of related species throughout embryogenesis. Recently, quantifiable approaches combining transcriptomic and evolutionary information provided novel evidence for the presence of a phylotranscriptomic hourglass pattern across kingdoms. As its biological function is unknown it remains speculative whether this pattern is functional or merely represents a nonfunctional evolutionary relic. The latter would seriously hamper future experimental approaches designed to test hypotheses regarding its function. Here, we address this question by generating transcriptome divergence index (TDI) profiles across embryogenesis of Danio rerio, Drosophila melanogaster, and Arabidopsis thaliana. To enable meaningful evaluation of the resulting patterns, we develop a statistical test that specifically assesses potential hourglass patterns. Based on this objective measure we find that two of these profiles follow a statistically significant hourglass pattern with the most conserved transcriptomes in the phylotypic periods. As the TDI considers only recent evolutionary signals, this indicates that the phylotranscriptomic hourglass pattern is not a rudiment but possibly actively maintained, implicating the existence of some linked biological function associated with embryogenesis in extant species. PMID:25631928

  10. Essential role of the TFIID subunit TAF4 in murine embryogenesis and embryonic stem cell differentiation.

    PubMed

    Langer, Diana; Martianov, Igor; Alpern, Daniel; Rhinn, Muriel; Keime, Céline; Dollé, Pascal; Mengus, Gabrielle; Davidson, Irwin

    2016-01-01

    TAF4 (TATA-binding protein-associated factor 4) and its paralogue TAF4b are components of the TFIID core module. We inactivated the murine Taf4a gene to address Taf4 function during embryogenesis. Here we show that Taf4a(-/-) embryos survive until E9.5 where primary germ layers and many embryonic structures are identified showing Taf4 is dispensable for their specification. In contrast, Taf4 is required for correct patterning of the trunk and anterior structures, ventral morphogenesis and proper heart positioning. Overlapping expression of Taf4a and Taf4b during embryogenesis suggests their redundancy at early stages. In agreement with this, Taf4a(-/-) embryonic stem cells (ESCs) are viable and comprise Taf4b-containing TFIID. Nevertheless, Taf4a(-/-) ESCs do not complete differentiation into glutamatergic neurons and cardiomyocytes in vitro due to impaired preinitiation complex formation at the promoters of critical differentiation genes. We define an essential role of a core TFIID TAF in differentiation events during mammalian embryogenesis. PMID:27026076

  11. Essential role of the TFIID subunit TAF4 in murine embryogenesis and embryonic stem cell differentiation

    PubMed Central

    Langer, Diana; Martianov, Igor; Alpern, Daniel; Rhinn, Muriel; Keime, Céline; Dollé, Pascal; Mengus, Gabrielle; Davidson, Irwin

    2016-01-01

    TAF4 (TATA-binding protein-associated factor 4) and its paralogue TAF4b are components of the TFIID core module. We inactivated the murine Taf4a gene to address Taf4 function during embryogenesis. Here we show that Taf4a−/− embryos survive until E9.5 where primary germ layers and many embryonic structures are identified showing Taf4 is dispensable for their specification. In contrast, Taf4 is required for correct patterning of the trunk and anterior structures, ventral morphogenesis and proper heart positioning. Overlapping expression of Taf4a and Taf4b during embryogenesis suggests their redundancy at early stages. In agreement with this, Taf4a−/− embryonic stem cells (ESCs) are viable and comprise Taf4b-containing TFIID. Nevertheless, Taf4a−/− ESCs do not complete differentiation into glutamatergic neurons and cardiomyocytes in vitro due to impaired preinitiation complex formation at the promoters of critical differentiation genes. We define an essential role of a core TFIID TAF in differentiation events during mammalian embryogenesis. PMID:27026076

  12. A novel role for vitamin K1 in a tyrosine phosphorylation cascade during chick embryogenesis.

    PubMed

    Saxena, S P; Fan, T; Li, M; Israels, E D; Israels, L G

    1997-02-15

    The development of the embryo is dependent upon a highly coordinated repertoire of cell division, differentiation, and migration. Protein-tyrosine phosphorylation plays a pivotal role in the regulation of these processes. Vitamin K-dependent gamma-carboxylated proteins have been identified as ligands for a unique family (Tyro 3 and 7) of receptor tyrosine kinases (RTKs) with transforming ability. The involvement of vitamin K metabolism and function in two well characterized birth defects, warfarin embryopathy and vitamin K epoxide reductase deficiency, suggests that developmental signals from K-dependent pathways may be required for normal embryogenesis. Using a chick embryogenesis model, we now demonstrate the existence of a vitamin K1-dependent protein-tyrosine phosphorylation cascade involving c-Eyk, a member of the Tyro 12 family, and key intracellular proteins, including focal adhesion kinase (pp125FAK), paxillin, and pp60src. This cascade is sensitive to alteration in levels or metabolism of vitamin K1. These findings provide a major clue as to why, in the mammalian (and human) fetus, the K-dependent proteins are maintained in an undercarboxylated state, even to the point of placing the newborn at hemorrhagic risk. The precise regulation of vitamin K1-dependent regulatory pathways would appear to be critical for orderly embryogenesis. PMID:9045861

  13. Key roles of the Broad-Complex gene in insect embryogenesis.

    PubMed

    Piulachs, Maria-Dolors; Pagone, Viviana; Bellés, Xavier

    2010-06-01

    The Broad-Complex (BR-C) is a primary response gene in the ecdysteroid signaling pathway which encodes a family of transcription factors that play a key role in metamorphic processes. With the aim of studying the possible functions of BR-C in the embryogenesis of a short germ band insect, we cloned BR-C cDNAs in the cockroach Blattella germanica (BgBR-C) and obtained the full coding region of six BR-C isoforms (Z1-Z6) of which, Z1-Z5 have homologous sequences in other insects, whereas Z6 is new. Temporal-expression patterns indicate that BgBR-C isoforms are present throughout the embryogenesis of B. germanica, although with weak fluctuations. Silencing all BgBR-C isoforms in the embryo through parental RNAi elicited a diversity of phenotypes. These phenotypes suggest roles for BgBR-C in different embryogenesis processes of B. germanica, mainly in the formation of the germ band, in the transition from 30% to 35% of total development, when the amnion and serosa rupture and the secondary dorsal organ is formed, and in the hatching process, which involves neural, endocrine and muscular coordination. PMID:20403438

  14. Nuclear lamins and peripheral nuclear antigens during fertilization and embryogenesis in mice and sea urchins

    NASA Technical Reports Server (NTRS)

    Schatten, G.; Schatten, H.; Simerly, C.; Maul, G. G.; Chaly, N.

    1985-01-01

    Nuclear structural changes during fertilization and embryogenesis in mice and sea urchins are traced using four antibodies. The oocytes from virgin female mice, morulae and blastocytes from mated females, and gametes from the sea urchin Lytechnius variegatis are studied using mouse monoclonal antibodies to nuclear lamin A/C, monoclonal antibody to P1, human autoimmune antibodies to lamin A/C, and to lamin B. The mouse fertilization data reveal no lamins on the oocyte; however, lamins are present on the pronuclei, and chromosomes are found on the oocytes and pronuclei. It is detected that on the sea urchin sperm the lamins are reduced to acrosomal and centriolar fossae and peripheral antigens are around the sperm nucleus. The mouse sperm bind lamin antibodies regionally and do not contain antigens. Lamins and antigens are observed on both pronuclei and chromosomes during sea urchin fertilization. Mouse embryogenesis reveals that lamin A/C is not recognized at morula and blastocyst stages; however, lamin B stains are retained. In sea urchin embryogenesis lamin recognition is lost at the blastrula, gastrula, and plutei stages. It is noted that nuclear lamins lost during spermatogenesis are restored at fertilization and peripheral antigens are associated with the surface of chromosomes during meiosis and mitosis and with the periphery of the pronuclei and nuclei during interphase.

  15. Cerebral Cavernous Malformations: Somatic Mutations in Vascular Endothelial Cells

    PubMed Central

    Gault, Judith; Awad, Issam A.; Recksiek, Peter; Shenkar, Robert; Breeze, Robert; Handler, Michael; Kleinschmidt-DeMasters, Bette Kay

    2009-01-01

    OBJECTIVE Germline mutations in three genes have been found in familial cases of cerebral cavernous malformations (CCM). We previously discovered somatic and germline truncating mutations in the KRIT1 gene supporting the “two-hit” mechanism of CCM lesion formation in a single lesion. The purpose of this study was to screen for somatic, nonheritable, mutations in three more lesions from different patients and identify the cell type(s) in which somatic mutations occur. METHODS Somatic mutations were sought in DNA from three surgically excised, fresh-frozen CCM lesions by cloning and screening PCR products generated from KRIT1 or PDCD10 coding regions. Laser capture microdissection (LCM) was used to isolated endothelial and nonendothelial cells in order to determine if somatic mutations were found in endothelial cells. RESULTS A CCM lesion harbored somatic and germline KRIT1 mutations on different chromosomes and are therefore biallelic. Both mutations are predicted to truncate the protein. The KRIT1 somatic mutations (novel c.1800delG mutation and previously identified 34 nucleotide deletion) in CCMs from two different patients were only found in the vascular endothelial cells lining caverns. No obvious somatic mutations were identified in the two other lesions; however, the results were inconclusive possibly due to the technical limitations or the fact that these specimens had a small proportion of vascular endothelial cells lining pristine caverns. CONCLUSION The “two-hit” mechanism occurs in vascular endothelial cells lining CCM caverns from two patients with somatic and Hispanic-American KRIT1 germline mutations. Methods for somatic mutation detection should focus on vascular endothelial cells lining pristine caverns. PMID:19574835

  16. SomaticSniper: identification of somatic point mutations in whole genome sequencing data

    PubMed Central

    Larson, David E.; Harris, Christopher C.; Chen, Ken; Koboldt, Daniel C.; Abbott, Travis E.; Dooling, David J.; Ley, Timothy J.; Mardis, Elaine R.; Wilson, Richard K.; Ding, Li

    2012-01-01

    Motivation: The sequencing of tumors and their matched normals is frequently used to study the genetic composition of cancer. Despite this fact, there remains a dearth of available software tools designed to compare sequences in pairs of samples and identify sites that are likely to be unique to one sample. Results: In this article, we describe the mathematical basis of our SomaticSniper software for comparing tumor and normal pairs. We estimate its sensitivity and precision, and present several common sources of error resulting in miscalls. Availability and implementation: Binaries are freely available for download at http://gmt.genome.wustl.edu/somatic-sniper/current/, implemented in C and supported on Linux and Mac OS X. Contact: delarson@wustl.edu; lding@wustl.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:22155872

  17. How Somatic Adult Tissues Develop Organizer Activity.

    PubMed

    Vogg, Matthias C; Wenger, Yvan; Galliot, Brigitte

    2016-01-01

    The growth and patterning of anatomical structures from specific cellular fields in developing organisms relies on organizing centers that instruct surrounding cells to modify their behavior, namely migration, proliferation, and differentiation. We discuss here how organizers can form in adult organisms, a process of utmost interest for regenerative medicine. Animals like Hydra and planarians, which maintain their shape and fitness thanks to a highly dynamic homeostasis, offer a useful paradigm to study adult organizers in steady-state conditions. Beside the homeostatic context, these model systems also offer the possibility to study how organizers form de novo from somatic adult tissues. Both extracellular matrix remodeling and caspase activation play a key role in this transition, acting as promoters of organizer formation in the vicinity of the wound. Their respective roles and the crosstalk between them just start to be deciphered. PMID:26970630

  18. The somatic shunt cable model for neurons.

    PubMed

    Durand, D

    1984-11-01

    The derivation of the equations for an electrical model of nerve cells is presented. The model consists of an equivalent cylinder, a lumped somatic impedance, and a variable shunt at the soma. This shunt was introduced to take into account the fast voltage decays observed following the injections of current pulses in some motoneurons and hippocampal granule cells that could not be explained by existing models. The shunt can be interpreted either by penetration damage with the electrode or by a lower membrane specific resistance at the soma than in the dendrites. A solution of the model equations is presented that allows the estimation of the electrotonic length L, the membrane time constant tau m, the dendritic dominance ratio rho, and the shunt parameter epsilon, based only on the measurement of the first two coefficients and time constants in the multiexponential voltage response to injected current pulses. PMID:6498277

  19. The Somatic Genomic Landscape of Glioblastoma

    PubMed Central

    Brennan, Cameron W.; Verhaak, Roel G.W.; McKenna, Aaron; Campos, Benito; Noushmehr, Houtan; Salama, Sofie R.; Zheng, Siyuan; Chakravarty, Debyani; Sanborn, J. Zachary; Berman, Samuel H.; Beroukhim, Rameen; Bernard, Brady; Wu, Chang-Jiun; Genovese, Giannicola; Shmulevich, Ilya; Barnholtz-Sloan, Jill; Zou, Lihua; Vegesna, Rahulsimham; Shukla, Sachet A.; Ciriello, Giovanni; Yung, WK; Zhang, Wei; Sougnez, Carrie; Mikkelsen, Tom; Aldape, Kenneth; Bigner, Darell D.; Van Meir, Erwin G.; Prados, Michael; Sloan, Andrew; Black, Keith L.; Eschbacher, Jennifer; Finocchiaro, Gaetano; Friedman, William; Andrews, David W.; Guha, Abhijit; Iacocca, Mary; O’Neill, Brian P.; Foltz, Greg; Myers, Jerome; Weisenberger, Daniel J.; Penny, Robert; Kucherlapati, Raju; Perou, Charles M.; Hayes, D. Neil; Gibbs, Richard; Marra, Marco; Mills, Gordon B.; Lander, Eric; Spellman, Paul; Wilson, Richard; Sander, Chris; Weinstein, John; Meyerson, Matthew; Gabriel, Stacey; Laird, Peter W.; Haussler, David; Getz, Gad; Chin, Lynda

    2013-01-01

    We describe the landscape of somatic genomic alterations based on multi-dimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer. PMID:24120142

  20. Cognitive Mechanisms Reciprocally Transmit Vulnerability between Depressive and Somatic Symptoms

    PubMed Central

    Harding, Kaitlin A.; Murphy, Karly M.

    2015-01-01

    Despite high comorbidity between depressive and somatic symptoms, cognitive mechanisms that transmit vulnerability between symptom clusters are largely unknown. Dampening, positive rumination, and brooding are three cognitive predictors of depression, with rumination theoretically indicated as a transdiagnostic vulnerability through amplifying and diminishing affect in response to events. Specifically, the excess negative affect and lack of positive affect characteristic of depressive symptoms and underlying somatic symptoms may cause and be caused by cognitive responses to events. Therefore, the current study examined whether comorbidity between depressive and somatic symptoms may be explained by the cognitive mechanisms of dampening and positive rumination in response to positive events and brooding in response to negative events among adults (N = 321) across eight weeks of assessment. We hypothesized that greater dampening and brooding would reciprocally predict greater depressive and somatic symptoms, while greater positive rumination would reciprocally predict fewer depressive and somatic symptoms. Mediation analyses in AMOS 22 indicated that dampening and brooding mediated reciprocal pathways between depressive and somatic symptoms, but positive rumination did not. Findings propose dampening and brooding as mechanisms of the reciprocal relationship between depressive and somatic symptoms through diminishing positive affect and amplifying negative affect in response to positive and negative events. PMID:26783455

  1. Somatic hypermutation maintains antibody thermodynamic stability during affinity maturation.

    PubMed

    Wang, Feng; Sen, Shiladitya; Zhang, Yong; Ahmad, Insha; Zhu, Xueyong; Wilson, Ian A; Smider, Vaughn V; Magliery, Thomas J; Schultz, Peter G

    2013-03-12

    Somatic hypermutation and clonal selection lead to B cells expressing high-affinity antibodies. Here we show that somatic mutations not only play a critical role in antigen binding, they also affect the thermodynamic stability of the antibody molecule. Somatic mutations directly involved in antigen recognition by antibody 93F3, which binds a relatively small hapten, reduce the melting temperature compared with its germ-line precursor by up to 9 °C. The destabilizing effects of these mutations are compensated by additional somatic mutations located on surface loops distal to the antigen binding site. Similarly, somatic mutations enhance both the affinity and thermodynamic stability of antibody OKT3, which binds the large protein antigen CD3. Analysis of the crystal structures of 93F3 and OKT3 indicates that these somatic mutations modulate antibody stability primarily through the interface of the heavy and light chain variable domains. The historical view of antibody maturation has been that somatic hypermutation and subsequent clonal selection increase antigen-antibody specificity and binding energy. Our results suggest that this process also optimizes protein stability, and that many peripheral mutations that were considered to be neutral are required to offset deleterious effects of mutations that increase affinity. Thus, the immunological evolution of antibodies recapitulates on a much shorter timescale the natural evolution of enzymes in which function and thermodynamic stability are simultaneously enhanced through mutation and selection. PMID:23440204

  2. An iTRAQ-Based Proteomics Approach to Clarify the Molecular Physiology of Somatic Embryo Development in Prince Rupprecht's Larch (Larix principis-rupprechtii Mayr)

    PubMed Central

    Zhao, Jian; Li, Hui; Fu, Shuangbin; Chen, Bo; Sun, Wenting; Zhang, Junqi; Zhang, Jinfeng

    2015-01-01

    Prince Rupprecht's larch (Larix principis-rupprechtii Mayr) is a native high-value forest tree species in North China whose clonal propagation through somatic embryogenesis (SE) has the potential to rapidly capture the benefits of breeding or genetic engineering programs and to improve raw material uniformity and quality. To date, research has focused on clarifying the molecular mechanism of SE, but proteomic studies are still in the early stages. In this study, isobaric tags for relative and absolute quantitation (iTRAQ) analysis was performed on three developmental stages of SE in L. principis-rupprechtii in an attempt to identify a wide range of proteins that are regulated differentially during this process. Proteins were extracted and analyzed from the pro-embryogenic mass (PEM), globular embryo (GE), and cotyledon embryo (CE) stages of embryo development. We detected 503 proteins in total and identified 96 proteins expressed differentially during different developmental stages. The identified proteins were analyzed further to provide information about their expression patterns and functions during SE. Four clusters of proteins based on shared expression profiles were generated. Functional analysis showed that proteins involved in primary metabolism, phosphorylation, and oxidation reduction were upregulated during somatic embryo development. This work provides novel insights into the process of larch embryo development in vitro and a basis for further study of the biological process and opportunities for practical application of this knowledge. PMID:25781987

  3. Auxin Biosynthesis, Accumulation, Action and Transport are Involved in Stress-Induced Microspore Embryogenesis Initiation and Progression in Brassica napus.

    PubMed

    Rodríguez-Sanz, Héctor; Solís, María-Teresa; López, María-Fernanda; Gómez-Cadenas, Aurelio; Risueño, María C; Testillano, Pilar S

    2015-07-01

    Isolated microspores are reprogrammed in vitro by stress, becoming totipotent cells and producing embryos and plants via a process known as microspore embryogenesis. Despite the abundance of data on auxin involvement in plant development and embryogenesis, no data are available regarding the dynamics of auxin concentration, cellular localization and the expression of biosynthesis genes during microspore embryogenesis. This work involved the analysis of auxin concentration and cellular accumulation; expression of TAA1 and NIT2 encoding enzymes of two auxin biosynthetic pathways; expression of the PIN1-like efflux carrier; and the effects of inhibition of auxin transport and action by N-1-naphthylphthalamic acid (NPA) and α-(p-chlorophenoxy) isobutyric acid (PCIB) during Brassica napus microspore embryogenesis. The results indicated de novo auxin synthesis after stress-induced microspore reprogramming and embryogenesis initiation, accompanying the first cell divisions. The progressive increase of auxin concentration during progression of embryogenesis correlated with the expression patterns of TAA1 and NIT2 genes of auxin biosynthetic pathways. Auxin was evenly distributed in early embryos, whereas in heart/torpedo embryos auxin was accumulated in apical and basal embryo regions. Auxin efflux carrier PIN1-like gene expression was induced in early multicellular embryos and increased at the globular/torpedo embryo stages. Inhibition of polar auxin transport (PAT) and action, by NPA and PCIB, impaired embryo development, indicating that PAT and auxin action are required for microspore embryo progression. NPA also modified auxin embryo accumulation patterns. These findings indicate that endogenous auxin biosynthesis, action and polar transport are required in stress-induced microspore reprogramming, embryogenesis initiation and progression. PMID:25907568

  4. Somatic mitochondrial DNA mutations in Chinese patients with osteosarcoma.

    PubMed

    Yu, Man; Wan, Yanfang; Zou, Qinghua

    2013-04-01

    Somatic mutations in mitochondrial DNA (mtDNA) have been long proposed to drive the pathogenesis and progression of human malignancies. Previous investigations have revealed a high frequency of somatic mutations in the D-loop control region of mtDNA in osteosarcoma. However, little is known with regard to whether or not somatic mutations also occur in the coding regions of mtDNA in osteosarcoma. To test this possibility, in the present study we screened somatic mutations over the full-length mitochondrial genome of 31 osteosarcoma tumour tissue samples, and corresponding peripheral blood samples from the same cohort of patients. We detected a sum of 11 somatic mutations in the mtDNA coding regions in our series. Nine of them were missense or frameshift mutations that have the potential to hamper mitochondrial respiratory function. In combination with our earlier observations on the D-loop fragment, 71.0% (22/31) of patients with osteosarcoma carried at least one somatic mtDNA mutation, and a total of 40 somatic mutations were identified. Amongst them, 29 (72.5%) were located in the D-loop region, two (5%) were in the sequences of the tRNA genes, two (5%) were in the mitochondrial ATP synthase subunit 6 gene and seven (17.5%) occurred in genes encoding components of the mitochondrial respiratory complexes. In addition, somatic mtDNA mutation was not closely associated with the clinicopathological characteristics of osteosarcoma. Together, these findings suggest that somatic mutations are highly prevalent events in both coding and non-coding regions of mtDNA in osteosarcoma. Some missense and frameshift mutations are putatively harmful to proper mitochondrial activity and might play vital roles in osteosarcoma carcinogenesis. PMID:23441585

  5. A somatic T15091C mutation in the Cytb gene of mouse mitochondrial DNA dominantly induces respiration defects.

    PubMed

    Hayashi, Chisato; Takibuchi, Gaku; Shimizu, Akinori; Mito, Takayuki; Ishikawa, Kaori; Nakada, Kazuto; Hayashi, Jun-Ichi

    2015-08-01

    Our previous studies provided evidence that mammalian mitochondrial DNA (mtDNA) mutations that cause mitochondrial respiration defects behave in a recessive manner, because the induction of respiration defects could be prevented with the help of a small proportion (10%-20%) of mtDNA without the mutations. However, subsequent studies found the induction of respiration defects by the accelerated accumulation of a small proportion of mtDNA with various somatic mutations, indicating the presence of mtDNA mutations that behave in a dominant manner. Here, to provide the evidence for the presence of dominant mutations in mtDNA, we used mouse lung carcinoma P29 cells and examined whether some mtDNA molecules possess somatic mutations that dominantly induce respiration defects. Cloning and sequence analysis of 40-48 mtDNA molecules from P29 cells was carried out to screen for somatic mutations in protein-coding genes, because mutations in these genes could dominantly regulate respiration defects by formation of abnormal polypeptides. We found 108 missense mutations existing in one or more of 40-48 mtDNA molecules. Of these missense mutations, a T15091C mutation in the Cytb gene was expected to be pathogenic due to the presence of its orthologous mutation in mtDNA from a patient with cardiomyopathy. After isolation of many subclones from parental P29 cells, we obtained subclones with various proportions of T15091C mtDNA, and showed that the respiration defects were induced in a subclone with only 49% T15091C mtDNA. Because the induction of respiration defects could not be prevented with the help of the remaining 51% mtDNA without the T15091C mutation, the results indicate that the T15091C mutation in mtDNA dominantly induced the respiration defects. PMID:26072375

  6. Neuronal correlates of symptom formation in functional somatic syndromes: a fMRI study.

    PubMed

    Landgrebe, Michael; Barta, Winfried; Rosengarth, Katharina; Frick, Ulrich; Hauser, Simone; Langguth, Berthold; Rutschmann, Roland; Greenlee, Mark W; Hajak, Goeran; Eichhammer, Peter

    2008-07-15

    Functional somatic syndromes are characterized by high morbidity due to various, fluctuating symptoms without objective somatic findings. There is increasing evidence for the contribution of emotional and cognitive functions to symptom formation, which has been well established in the perception of pain. In addition to their involvement in various other cognitive and emotional processes, the anterior cingulate and insular cortex are thought to contribute to the so-called "pain neuromatrix". Recent data suggest that these areas appear also to be involved in symptom manifestation in multiple chemical sensitivity. Here we used functional Magnetic Resonance Imaging (fMRI) to test whether this network is also involved in the induction of unpleasant perceptions by sham mobile phone radiation in subjectively electrosensitive patients. This design enabled us to completely dissociate the unpleasant subjective perception from any real physical stimulus. Fifteen subjectively electrosensitive patients and 15 age- and gender-matched healthy controls were exposed to sham mobile phone radiation and heat as a control condition. The perceived stimulus intensities were rated on a five-point scale. During anticipation of and exposure to sham mobile phone radiation increased activations in anterior cingulate and insular cortex as well as fusiform gyrus were seen in the electrosensitive group compared to controls, while heat stimulation led to similar activations in both groups. Symptom manifestation during sham exposure to mobile phone radiation was accompanied by specific alterations of cortical activity in anterior cingulate and insular cortex in subjectively electrosensitive patients further supporting the involvement of these areas in the perception of unpleasantness and generation of functional somatic syndromes. PMID:18499479

  7. Cotyledonary somatic embryos of Pinus pinaster Ait. most closely resemble fresh, maturing cotyledonary zygotic embryos: biological, carbohydrate and proteomic analyses.

    PubMed

    Morel, Alexandre; Trontin, Jean-François; Corbineau, Françoise; Lomenech, Anne-Marie; Beaufour, Martine; Reymond, Isabelle; Le Metté, Claire; Ader, Kevin; Harvengt, Luc; Cadene, Martine; Label, Philippe; Teyssier, Caroline; Lelu-Walter, Marie-Anne

    2014-11-01

    Cotyledonary somatic embryos (SEs) of maritime pine are routinely matured for 12 weeks before being germinated and converted to plantlets. Although regeneration success is highly dependent on SEs quality, the date of harvesting is currently determined mainly on the basis of morphological features. This empirical method does not provide any accurate information about embryo quality with respect to storage compounds (proteins, carbohydrates). We first analyzed SEs matured for 10, 12 and 14 weeks by carrying out biological (dry weight, water content) and biochemical measurements (total protein and carbohydrate contents). No difference could be found between collection dates, suggesting that harvesting SEs after 12 weeks is appropriate. Cotyledonary SEs were then compared to various stages, from fresh to fully desiccated, in the development of cotyledonary zygotic embryos (ZEs). We identified profiles that were similar using hierarchical ascendant cluster analysis (HCA). Fresh and dehydrated ZEs could be distinguished, and SEs clustered with fresh ZEs. Both types of embryo exhibited similar carbohydrate and protein contents and signatures. This high level of similarity (94.5 %) was further supported by proteome profiling. Highly expressed proteins included storage, stress-related, late embryogenesis abundant and energy metabolism proteins. By comparing overexpressed proteins in developing and cotyledonary SEs or ZEs, some (23 proteins) could be identified as candidate biomarkers for the late, cotyledonary stage. This is the first report of useful generic protein markers for monitoring embryo development in maritime pine. Our results also suggest that improvements of SEs quality may be achieved if the current maturation conditions are refined. PMID:25115559

  8. Proteome Analysis Unravels Mechanism Underling the Embryogenesis of the Honeybee Drone and Its Divergence with the Worker (Apis mellifera lingustica).

    PubMed

    Fang, Yu; Feng, Mao; Han, Bin; Qi, Yuping; Hu, Han; Fan, Pei; Huo, Xinmei; Meng, Lifeng; Li, Jianke

    2015-09-01

    The worker and drone bees each contain a separate diploid and haploid genetic makeup, respectively. Mechanisms regulating the embryogenesis of the drone and its mechanistic difference with the worker are still poorly understood. The proteomes of the two embryos at three time-points throughout development were analyzed by applying mass spectrometry-based proteomics. We identified 2788 and 2840 proteins in the worker and drone embryos, respectively. The age-dependent proteome driving the drone embryogenesis generally follows the worker's. The two embryos however evolve a distinct proteome setting to prime their respective embryogenesis. The strongly expressed proteins and pathways related to transcriptional-translational machinery and morphogenesis at 24 h drone embryo relative to the worker, illustrating the earlier occurrence of morphogenesis in the drone than worker. These morphogenesis differences remain through to the middle-late stage in the two embryos. The two embryos employ distinct antioxidant mechanisms coinciding with the temporal-difference organogenesis. The drone embryo's strongly expressed cytoskeletal proteins signify key roles to match its large body size. The RNAi induced knockdown of the ribosomal protein offers evidence for the functional investigation of gene regulating of honeybee embryogenesis. The data significantly expand novel regulatory mechanisms governing the embryogenesis, which is potentially important for honeybee and other insects. PMID:26260241

  9. Consequences of the recurrent MYD88L265P somatic mutation for B cell tolerance

    PubMed Central

    Wang, James Q.; Jeelall, Yogesh S.; Beutler, Bruce

    2014-01-01

    MYD88L265P has recently been discovered as an extraordinarily frequent somatic mutation in benign monoclonal IgM gammopathy, Waldenström’s macroglobulinemia, and diffuse large B cell lymphoma. In this study, we analyze the consequences for antigen-activated primary B cells of acquiring MYD88L265P. The mutation induced rapid B cell division in the absence of exogenous TLR ligands and was inhibited by Unc93b13d mutation and chloroquine or TLR9 deficiency, indicating continued dependence on upstream TLR9 activation. Proliferation and NF-κB activation induced by MYD88L265P were nevertheless rapidly countered by the induction of TNFAIP3, an NF-κB inhibitor frequently inactivated in MYD88L265P–bearing lymphomas, and extinguished by Bim-dependent apoptosis. MYD88L265P caused self-reactive B cells to accumulate in vivo only when apoptosis was opposed by Bcl2 overexpression. These results reveal checkpoints that fortify TLR responses against aberrant B cell proliferation in response to ubiquitous TLR and BCR self-ligands and suggest that tolerance failure requires the accumulation of multiple somatic mutations. PMID:24534189

  10. Calcineurin-NFAT Signaling Controls Somatic Cell Reprogramming in a Stage-Dependent Manner.

    PubMed

    Sun, Ming; Liao, Bing; Tao, Yu; Chen, Hao; Xiao, Feng; Gu, Junjie; Gao, Shaorong; Jin, Ying

    2016-05-01

    Calcineurin-NFAT signaling is critical for early lineage specification of mouse embryonic stem cells and early embryos. However, its roles in somatic cell reprogramming remain unknown. Here, we report that calcineurin-NFAT signaling has a dynamic activity and plays diverse roles at different stages of reprogramming. At the early stage, calcineurin-NFAT signaling is transiently activated and its activation is required for successful reprogramming. However, at the late stage of reprogramming, activation of calcineurin-NFAT signaling becomes a barrier for reprogramming and its inactivation is critical for successful induction of pluripotency. Mechanistically, calcineurin-NFAT signaling contributes to the reprogramming through regulating multiple early events during reprogramming, including mesenchymal to epithelial transition (MET), cell adhesion and emergence of SSEA1(+) intermediate cells. Collectively, this study reveals for the first time the important roles of calcineurin-NFAT signaling during somatic cell reprogramming and provides new insights into the molecular regulation of reprogramming. J. Cell. Physiol. 231: 1151-1162, 2016. © 2015 Wiley Periodicals, Inc. PMID:26448199

  11. Constitutive heterochromatin reorganization during somatic cell reprogramming

    PubMed Central

    Fussner, Eden; Djuric, Ugljesa; Strauss, Mike; Hotta, Akitsu; Perez-Iratxeta, Carolina; Lanner, Fredrik; Dilworth, F Jeffrey; Ellis, James; Bazett-Jones, David P

    2011-01-01

    Induced pluripotent stem (iPS) cell reprogramming is a gradual epigenetic process that reactivates the pluripotent transcriptional network by erasing and establishing repressive epigenetic marks. In contrast to loci-specific epigenetic changes, heterochromatin domains undergo epigenetic resetting during the reprogramming process, but the effect on the heterochromatin ultrastructure is not known. Here, we characterize the physical structure of heterochromatin domains in full and partial mouse iPS cells by correlative electron spectroscopic imaging. In somatic and partial iPS cells, constitutive heterochromatin marked by H3K9me3 is highly compartmentalized into chromocentre structures of densely packed chromatin fibres. In contrast, chromocentre boundaries are poorly defined in pluripotent embryonic stem and full iPS cells, and are characterized by unusually dispersed 10 nm heterochromatin fibres in high Nanog-expressing cells, including pluripotent cells of the mouse blastocyst before differentiation. This heterochromatin reorganization accompanies retroviral silencing during conversion of partial iPS cells by MEK/GSK3 2i inhibitor treatment. Thus, constitutive heterochromatin is compacted in partial iPS cells but reorganizes into dispersed 10 nm chromatin fibres as the fully reprogrammed iPS cell state is acquired. PMID:21468033

  12. Dysphoria and somatization in Iranian culture.

    PubMed Central

    Pliskin, K L

    1992-01-01

    Iranians express dysphoria through an undifferentiated term called narahati, meaning depressed, ill at ease, nervous, inconvenienced, or anxious. People try masking this emotion or express it in specific ways nonverbally, such as sulking or not eating. Two other dysphoric affects, sadness and anger, are not masked. Because of the social conception of persons being emotionally sensitive, the expression of narahati is guarded: expressing it not only could show that one is socially vulnerable, it could also make another sensitive empathic person narahat. The body is also sensitive, but to the physical world. Physical health is maintained by balancing a diet of "hot" and "cold" foods and avoiding exposure to cold and moisture. With the social and cultural problems brought on by revolution, war, immigration, and accommodation to a new society, Iranian refugees experience changes in family, role, status, finances, language, and other sociocultural ways of being that cause them to feel narahat and to express it verbally, nonverbally, or through somatization. Understanding Iranian conceptions of emotional and physical sensitivity will help clinicians in treating Iranian patients. PMID:1413773

  13. Cloning animals by somatic cell nuclear transfer--biological factors.

    PubMed

    Tian, X Cindy; Kubota, Chikara; Enright, Brian; Yang, Xiangzhong

    2003-11-13

    Cloning by nuclear transfer using mammalian somatic cells has enormous potential application. However, somatic cloning has been inefficient in all species in which live clones have been produced. High abortion and fetal mortality rates are commonly observed. These developmental defects have been attributed to incomplete reprogramming of the somatic nuclei by the cloning process. Various strategies have been used to improve the efficiency of nuclear transfer, however, significant breakthroughs are yet to happen. In this review we will discuss studies conducted, in our laboratories and those of others, to gain a better understanding of nuclear reprogramming. Because cattle are a species widely used for nuclear transfer studies, and more laboratories have succeeded in cloning cattle than any other species, this review will be focused on somatic cell cloning of cattle. PMID:14614770

  14. The place of asymmetric somatic hybridization in wheat breeding.

    PubMed

    Liu, Shuwei; Xia, Guangmin

    2014-04-01

    Since its first development some 40 years ago, the application of the somatic hybridization technique has generated a body of hybrid plant material involving a wide combination of parental species. Until the late 1990s, the technique was ineffective in wheat, as regeneration from protoplasts was proving difficult to achieve. Since this time, however, a successful somatic hybridization protocol for wheat has been established and used to generate a substantial number of both symmetric and asymmetric somatic hybrids and derived materials, especially involving the parental combination bread wheat and tall wheatgrass (Thinopyrum ponticum). This review describes the current state of the art for somatic hybridization in wheat and focuses on its potential application for wheat improvement. PMID:24370665

  15. Production of goats by somatic cell nuclear transfer.

    PubMed

    Baguisi, A; Behboodi, E; Melican, D T; Pollock, J S; Destrempes, M M; Cammuso, C; Williams, J L; Nims, S D; Porter, C A; Midura, P; Palacios, M J; Ayres, S L; Denniston, R S; Hayes, M L; Ziomek, C A; Meade, H M; Godke, R A; Gavin, W G; Overström, E W; Echelard, Y

    1999-05-01

    In this study, we demonstrate the production of transgenic goats by nuclear transfer of fetal somatic cells. Donor karyoplasts were obtained from a primary fetal somatic cell line derived from a 40-day transgenic female fetus produced by artificial insemination of a nontransgenic adult female with semen from a transgenic male. Live offspring were produced with two nuclear transfer procedures. In one protocol, oocytes at the arrested metaphase II stage were enucleated, electrofused with donor somatic cells, and simultaneously activated. In the second protocol, activated in vivo oocytes were enucleated at the telophase II stage, electrofused with donor somatic cells, and simultaneously activated a second time to induce genome reactivation. Three healthy identical female offspring were born. Genotypic analyses confirmed that all cloned offspring were derived from the donor cell line. Analysis of the milk of one of the transgenic cloned animals showed high-level production of human antithrombin III, similar to the parental transgenic line. PMID:10331804

  16. Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer

    PubMed Central

    Tachibana, Masahito; Amato, Paula; Sparman, Michelle; Gutierrez, Nuria Marti; Tippner-Hedges, Rebecca; Ma, Hong; Kang, Eunju; Fulati, Alimujiang; Lee, Hyo-Sang; Sritanaudomchai, Hathaitip; Masterson, Keith; Larson, Janine; Eaton, Deborah; Sadler-Fredd, Karen; Battaglia, David; Lee, David; Wu, Diana; Jensen, Jeffrey; Patton, Phillip; Gokhale, Sumita; Stouffer, Richard L.; Wolf, Don; Mitalipov, Shoukhrat

    2013-01-01

    SUMMARY Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state. PMID:23683578

  17. Extensive load of somatic CNVs in the human placenta.

    PubMed

    Kasak, Laura; Rull, Kristiina; Vaas, Pille; Teesalu, Pille; Laan, Maris

    2015-01-01

    Placenta is a temporary, but indispensable organ in mammalian pregnancy. From its basic nature, it exhibits highly invasive tumour-like properties facilitating effective implantation through trophoblast cell proliferation and migration, and a critical role in pregnancy success. We hypothesized that similarly to cancer, somatic genomic rearrangements are promoted in the support of placental function. Here we present the first profiling of copy number variations (CNVs) in human placental genomes, showing an extensive load of somatic CNVs, especially duplications and suggesting that this phenomenon may be critical for normal gestation. Placental somatic CNVs were significantly enriched in genes involved in cell adhesion, immunity, embryonic development and cell cycle. Overrepresentation of imprinted genes in somatic duplications suggests that amplified gene copies may represent an alternative mechanism to support parent-of-origin specific gene expression. Placentas from pregnancy complications exhibited significantly altered CNV profile compared to normal gestations, indicative to the clinical implications of the study. PMID:25666259

  18. Embryo defective 14 encodes a plastid-targeted cGTPase essential for embryogenesis in maize.

    PubMed

    Li, Cuiling; Shen, Yun; Meeley, Robert; McCarty, Donald R; Tan, Bao-Cai

    2015-11-01

    The embryo defective (emb) mutants in maize genetically define a unique class of loci that is required for embryogenesis but not endosperm development, allowing dissection of two developmental processes of seed formation. Through characterization of the emb14 mutant, we report here that Emb14 gene encodes a circular permuted, YqeH class GTPase protein that likely functions in 30S ribosome formation in plastids. Loss of Emb14 function in the null mutant arrests embryogenesis at the early transition stage. Emb14 was cloned by transposon tagging and was confirmed by analysis of four alleles. Subcellular localization indicated that the EMB14 is targeted to chloroplasts. Recombinant EMB14 is shown to hydrolyze GTP in vitro (Km  = 2.42 ± 0.3 μm). Emb14 was constitutively expressed in all tissues examined and high level of expression was found in transition stage embryos. Comparison of emb14 and WT indicated that loss of EMB14 function severely impairs accumulation of 16S rRNA and several plastid encoded ribosomal genes. We show that an EMB14 transgene complements the pale green, slow growth phenotype conditioned by mutations in AtNOA1, a closely related YqeH GTPase of Arabidopsis. Taken together, we propose that the EMB14/AtNOA1/YqeH class GTPases function in assembly of the 30S subunit of the chloroplast ribosome, and that this function is essential to embryogenesis in plants. PMID:26771182

  19. The Genomic Distribution and Function of Histone Variant HTZ-1 during C. elegans Embryogenesis

    PubMed Central

    Whittle, Christina M.; McClinic, Karissa N.; Ercan, Sevinc; Zhang, Xinmin; Green, Roland D.; Kelly, William G.; Lieb, Jason D.

    2008-01-01

    In all eukaryotes, histone variants are incorporated into a subset of nucleosomes to create functionally specialized regions of chromatin. One such variant, H2A.Z, replaces histone H2A and is required for development and viability in all animals tested to date. However, the function of H2A.Z in development remains unclear. Here, we use ChIP-chip, genetic mutation, RNAi, and immunofluorescence microscopy to interrogate the function of H2A.Z (HTZ-1) during embryogenesis in Caenorhabditis elegans, a key model of metazoan development. We find that HTZ-1 is expressed in every cell of the developing embryo and is essential for normal development. The sites of HTZ-1 incorporation during embryogenesis reveal a genome wrought by developmental processes. HTZ-1 is incorporated upstream of 23% of C. elegans genes. While these genes tend to be required for development and occupied by RNA polymerase II, HTZ-1 incorporation does not specify a stereotypic transcription program. The data also provide evidence for unexpectedly widespread independent regulation of genes within operons during development; in 37% of operons, HTZ-1 is incorporated upstream of internally encoded genes. Fewer sites of HTZ-1 incorporation occur on the X chromosome relative to autosomes, which our data suggest is due to a paucity of developmentally important genes on X, rather than a direct function for HTZ-1 in dosage compensation. Our experiments indicate that HTZ-1 functions in establishing or maintaining an essential chromatin state at promoters regulated dynamically during C. elegans embryogenesis. PMID:18787694

  20. Developmental origins of neurotransmitter and transcriptome alterations in adult female zebrafish exposed to atrazine during embryogenesis.

    PubMed

    Wirbisky, Sara E; Weber, Gregory J; Sepúlveda, Maria S; Xiao, Changhe; Cannon, Jason R; Freeman, Jennifer L

    2015-07-01

    Atrazine is an herbicide applied to agricultural crops and is indicated to be an endocrine disruptor. Atrazine is frequently found to contaminate potable water supplies above the maximum contaminant level of 3μg/L as defined by the U.S. Environmental Protection Agency. The developmental origin of adult disease hypothesis suggests that toxicant exposure during development can increase the risk of certain diseases during adulthood. However, the molecular mechanisms underlying disease progression are still unknown. In this study, zebrafish embryos were exposed to 0, 0.3, 3, or 30μg/L atrazine throughout embryogenesis. Larvae were then allowed to mature under normal laboratory conditions with no further chemical treatment until 7 days post fertilization (dpf) or adulthood and neurotransmitter analysis completed. No significant alterations in neurotransmitter levels was observed at 7dpf or in adult males, but a significant decrease in 5-hydroxyindoleacetic acid (5-HIAA) and serotonin turnover was seen in adult female brain tissue. Transcriptomic analysis was completed on adult female brain tissue to identify molecular pathways underlying the observed neurological alterations. Altered expression of 1928, 89, and 435 genes in the females exposed to 0.3, 3, or 30μg/L atrazine during embryogenesis were identified, respectively. There was a high level of overlap between the biological processes and molecular pathways in which the altered genes were associated. Moreover, a subset of genes was down regulated throughout the serotonergic pathway. These results provide support of the developmental origins of neurological alterations observed in adult female zebrafish exposed to atrazine during embryogenesis. PMID:25929836

  1. Selection against somatic parasitism can maintain allorecognition in fungi.

    PubMed

    Czárán, Tamas; Hoekstra, Rolf F; Aanen, Duur K

    2014-12-01

    Fusion between multicellular individuals is possible in many organisms with modular, indeterminate growth, such as marine invertebrates and fungi. Although fusion may provide various benefits, fusion usually is restricted to close relatives by allorecognition, also called heterokaryon or somatic incompatibility in fungi. A possible selective explanation for allorecognition is protection against somatic parasites. Such mutants contribute less to colony functions but more to reproduction. However, previous models testing this idea have failed to explain the high diversity of allorecognition alleles in nature. These models did not, however, consider the possible role of spatial structure. We model the joint evolution of allorecognition and somatic parasitism in a multicellular organism resembling an asexual ascomycete fungus in a spatially explicit simulation. In a 1000-by-1000 grid, neighbouring individuals can fuse, but only if they have the same allotype. Fusion with a parasitic individual decreases the total reproductive output of the fused individuals, but the parasite compensates for this individual-level fitness reduction by a disproportional share of the offspring. Allorecognition prevents the invasion of somatic parasites, and vice versa, mutation towards somatic parasitism provides the selective conditions for extensive allorecognition diversity. On the one hand, if allorecognition diversity did not build up fast enough, somatic parasites went to fixation; conversely, once parasites had gone to fixation no allorecognition diversity built up. On the other hand, the mere threat of parasitism could select for high allorecognition diversity, preventing invasion of somatic parasites. Moderate population viscosity combined with weak global dispersal was optimal for the joint evolution of allorecognition and protection against parasitism. Our results are consistent with the widespread occurrence of allorecognition in fungi and the low degree of somatic parasitism. We discuss the implications of our results for allorecognition in other organism groups. PMID:25305337

  2. Involvement of amygdaloid corticosterone in altered visceral and somatic sensation.

    PubMed

    Myers, Brent; Dittmeyer, Kale; Greenwood-Van Meerveld, Beverley

    2007-07-19

    Behavioral responses to thermal and mechanical stimuli were used to examine somatic sensitivity in rats with stereotaxic corticosterone (CORT) implants on the amygdala. These animals have previously been shown to display anxiety-like behavior coupled with colonic hypersensitivity; in this study, CORT induced not only visceral hypersensitivity but also somatic hypersensitivity. These findings suggest that modulation of the amygdala with CORT results in a generalized decrease in sensory thresholds via descending neuronal pathways. PMID:17481745

  3. [Somatic mutations in nuclear and mitochondrial DNA]. Progress report

    SciTech Connect

    Not Available

    1992-09-01

    The study is concerned the design of new assays that may detect rare somatic mutations in nuclear and mitochondrial DNA, which may increase upon exposure to mutagens, and thus become a marker of human exposure to such mutagens. Two assays for somatic mutation were presented, one for mitochondrial DNA deletions which was developed by the author, and one for deletions of the ADA gene which resides in the nucleus.

  4. Somatization in the community: relationship to disability and use of services.

    PubMed Central

    Escobar, J I; Golding, J M; Hough, R L; Karno, M; Burnam, M A; Wells, K B

    1987-01-01

    We tested the hypotheses that an abridged somatization construct that we had developed would be associated with use of health services, preferential use of medical over mental health services, and an index of disability. These hypotheses were tested using structured interview data from 3,132 randomly selected community respondents. We found that: respondents meeting criteria for somatization reported a heavier use of health services than non-somatizers; of those respondents meeting criteria for a psychiatric diagnosis, somatizers preferentially used medical over mental health services whereas non-somatizers reported the opposite trend; and somatizers were more likely than non-somatizers to report recent sick leave or restricted activity. PMID:3592038

  5. A Chemical and Physical Characterization of Echinoid RNA during Early Embryogenesis

    PubMed Central

    Slater, D. W.; Spiegelman, S.

    1966-01-01

    The properties of the major RNA components prepared from Arbacia punctulata have been characterized by sedimentation in sucrose gradients and analysis of base composition. Comparison of the components found in the unfertilized egg with those observed subsequent to fertilization revealed no differences in any of the embryonic stages examined. The base composition and sedimentation profiles of RNA from unfertilized nuclear egg fragments and from 105,000 g pellet were similar to those of the intact egg. It is concluded that the early stages of embryogenesis are not accompanied by detectable alteration of the physical or chemical characteristics of the major RNA components found in the unfertilized egg. PMID:19210966

  6. Somatic symptoms, severe mood dysregulation, and aggressiveness in preschool children.

    PubMed

    Serra Giacobo, Rodrigo; Jan, Ma Claustre; Bonillo, Albert; Ballesp, Sergi; Daz-Regaon, Natalia

    2012-01-01

    Many researchers have studied somatic symptoms in children. However, its association with severe mood dysregulation (SMD) is poorly known. The aim of this study is to detect the presence of SMD in preschool children and to know the prevalence of somatic symptoms and associations with psychopathology, SMD, and aggressiveness. The study population consists of children between 3 to 6 years of age enrolled in Barcelona's kindergarten schools (n?=?319). Their parents completed questionnaires about the presence of somatic symptoms in children, absences from school and pediatric visits, child psychiatric symptoms, presence of symptoms of SMD, and aggressiveness. Teachers were also informed about SMD and aggressiveness. Children who complained frequent somatic symptoms (three or more in the last 2 weeks) were compared with those who did not. Two hundred five children (64.3%) reported at least one physical complaint in the 2 weeks preceding the study. One hundred participants (31.3%) reported frequent somatic complaints. Positive associations were found with anxiety symptomatology, separation anxiety, social phobia, pediatric visits, and school absences, but not with aggressiveness or SMD symptoms. Somatic symptoms are common in a sample of preschool children but do not show a positive association with the symptoms of SMD. PMID:21611729

  7. [Reactivity of microvessels in the cerebral hemispheres and the skeletal muscles in chicken during second half of embryogenesis].

    PubMed

    Belichenko, V M; Turganbaeva, A S; Shoshenko, K A

    2014-02-01

    At Leghorn hens in the second half of embryogenesis and in 4-day-old chicks are studied reaction volume flow velocity (VF) in the superficial layers of the cerebral hemispheres and in skeletal muscle (Lazer Doppler FIowmetry) after the local influence of norepinephrine and sodium nitroprusside. It is shown that the response to these substances begins to manifest itself in the hemisphere in the last quarter of embryogenesis and authentically expressed by the end of it and in the chickens. It is noted that the response to these substances skeletal muscle VF (according to the new and previously published data on gastrocnemius and pectoral muscle) is also clearly manifested by the end of embryogenesis. PMID:25470895

  8. Somatic mosaicism in plants with special reference to somatic crossing over

    PubMed Central

    Vig, Baldev K.

    1978-01-01

    Plant systems in use for the detection of environmental mutagens appear capable of detecting all types of genetic effects which can be studied in animals. The study of somatic mosaicism, however, is better developed in plants than in higher animals. A case is presented here which shows the ability of plant systems in analyzing a host of genetic end points, including chromosome aberrations like deletions, somatic crossing over, numerical inequality, gene conversion, paramutations and point mutations. The systems in general use utilize certain varieties of Tradescantia, Glycine max, Nicotiana tabacum, Antirrhinum majus, Petunia hybrida, and Arabidopsis thaliana. Heterozygous plants or their homozygous counterparts with gene markers affecting chlorophyll development or anthocyanin in floral parts are exploited in these studies. Mutagens produce different frequencies of different types of spots typical of the mode of action of the agent. Analysis of these parameters may be used to predict, at least qualitatively, the kind of genetic damage that might be produced in man. Besides, one can test the validity of interpretation by traditional progeny tests of plants raised from tissue culture from sectors as in Nicotiana and/or by precursor analysis as done in Antirrhinum. The study of mosaicism in plants offers quite inexpensive, rapid, and reliable tests of mutagenicity at least as a preliminary eukaryotic test system. ImagesFIGURE 1.FIGURE 1.FIGURE 2.FIGURE 9. PMID:367771

  9. Reprogramming of somatic cells induced by fusion of embryonic stem cells using hemagglutinating virus of Japan envelope (HVJ-E)

    SciTech Connect

    Yue, Xiao-shan; Department of Biomolecular Engineering, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501 ; Fujishiro, Masako; Toyoda, Masashi; Akaike, Toshihiro; Ito, Yoshihiro; Department of Biomolecular Engineering, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501

    2010-04-16

    In this research, hemagglutinating virus of Japan envelope (HVJ-E) was used to reprogram somatic cells by fusion with mouse embryonic stem (ES) cells. Neomycin-resistant mouse embryonic fibroblasts (MEFs) were used as somatic cells. Nanog-overexpressing puromycin-resistant EB3 cells were used as mouse ES cells. These two cells were fused by exposing to HVJ-E and the generated fusion cells were selected by puromycin and G418 to get the stable fusion cell line. The fusion cells form colonies in feeder-free culture system. Microsatellite analysis of the fusion cells showed that they possessed genes from both ES cells and fibroblasts. The fusion cells were tetraploid, had alkali phosphatase activity, and expressed stem cell marker genes such as Pou5f1, Nanog, and Sox2, but not the fibroblast cell marker genes such as Col1a1 and Col1a2. The pluripotency of fusion cells was confirmed by their expression of marker genes for all the three germ layers after differentiation induction, and by their ability to form teratoma which contained all the three primary layers. Our results show that HVJ-E can be used as a fusion reagent for reprogramming of somatic cells.

  10. In vitro IFN-gamma production by goat blood cells after stimulation with somatic and secreted Corynebacterium pseudotuberculosis antigens.

    PubMed

    Meyer, R; Regis, L; Vale, V; Paule, B; Carminati, R; Bahia, R; Moura-Costa, L; Schaer, R; Nascimento, I; Freire, S

    2005-09-15

    Corynebacterium pseudotuberculosis is the causal agent of caseous lymphadenitis, a chronic illness that attacks goats and sheep characterized by pyogranulomas formation in lymph nodes and organs. Regarding the current knowledge of the pathogenesis of the caseous lymphadenitis, there is evidence that besides the humoral response the induction of a durable cellular response is fundamental for its control. In this sense, research on antigens of C. pseudotuberculosis that are capable to inducing cellular immunity is an important step for the development of diagnosis tests and more efficient vaccines. In the present study, the interferon-gamma production in cultures of whole blood from infected goats stimulated with secreted bacterial antigen or somatic antigen were used to evaluate the cellular response. The results demonstrated a significant difference in the ability of the two antigens to induce a cellular response. That is, IFN-gamma production was high with cells from infected animals in response to the secreted antigen while IFN-gamma production was low when somatic antigen was used. The concomitant use of these antigens with PWM also showed differences. That is, the secreted antigen increased the IFN-gamma production induced by PWM, while the somatic antigen seems not to have altered the response to PWM. PMID:15982750

  11. Vagal stomach afferents inhibit somatic pain perception.

    PubMed

    Sedan, Oshra; Sprecher, Elliot; Yarnitsky, David

    2005-02-01

    Vagal stimulation inhibits systemic pain perception in animals, probably via the nucleus tractus solitarius and its connections with descending nuclei in the brainstem which inhibit pain. Pain-inhibiting effects of such stimulation in humans, obtained from epileptic patients treated by vagal stimulation, are controversial. The aim of our study was to evaluate whether vagal stomach afferent activation inhibits pain perception in healthy humans. Pain thresholds, magnitude of tonic heat pain at 46 degrees C stimulation, pain temporal summation and laser pain evoked potentials were measured at the hand before and immediately after rapid drinking of 1500 ml water in 31 volunteers. We found an increase in heat pain threshold from 43.3+/-2.6 to 44.7+/-2.2 degrees C, P<0.0001, a decrease of peak pain magnitude to tonic heat from 56.3+/-26.2 to 43.7+/-25.8 (on 0-100 VAS), P<0.0001, a lowering of area under the curve during tonic noxious heat stimulus from 1962+/-984 to 1411+/-934, P<0.001. Additionally, we observed a decrease in the peak to peak evoked potential amplitude from 19.2 microV+/-1.2 to 15.6 microV+/-1.2 (P=0.005) together with a decrease in the estimation of mean laser induced pain from 52.28+/-18.00 to 48.14+/-20.18 (P=0.025). Mechanical pain thresholds and temporal summation did not change significantly. We conclude that vagal stomach afferents exert an inhibitory effect on somatic pain perception in humans. PMID:15661444

  12. Electromagnetic induction methods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Electromagnetic induction geophysical methods are finding greater and greater use for agricultural purposes. Electromagnetic induction methods measure the electrical conductivity (or resistivity) for a bulk volume of soil directly beneath the surface. An instrument called a ground conductivity meter...

  13. The low molecular weight fraction of compounds released from immature wheat pistils supports barley pollen embryogenesis.

    PubMed

    Lippmann, Rico; Friedel, Swetlana; Mock, Hans-Peter; Kumlehn, Jochen

    2015-01-01

    Pollen embryogenesis provides a useful means of generating haploid plants for plant breeding and basic research. Although it is well-established that the efficacy of the process can be enhanced by the provision of immature pistils as a nurse tissue, the origin and compound class of the signal molecule(s) involved is still elusive. Here, a micro-culture system was established to enable the culturing of populations of barley pollen at a density too low to allow unaided embryogenesis to occur, and this was then exploited to assess the effect of using various parts of the pistil as nurse tissue. A five-fold increase in the number of embryogenic calli formed was obtained by simply cutting the pistils in half. The effectiveness of the pistil-conditioned medium was transitory, since it needed replacement at least every 4 days to measurably ensure embryogenic development. The differential effect of various size classes of compounds present in the pistil-conditioned medium showed that the relevant molecule(s) was of molecular weight below 3 kDa. This work narrows down possible feeder molecules to lower molecular weight compounds and showed that the cellular origin of the active compound(s) is not specific to any tested part of the pistil. Furthermore, the increased recovery of calli during treatment with cut pistils may provide a useful tool for plant breeders and researchers using haploid technology in barley and other plant species. PMID:26217352

  14. The low molecular weight fraction of compounds released from immature wheat pistils supports barley pollen embryogenesis

    PubMed Central

    Lippmann, Rico; Friedel, Swetlana; Mock, Hans-Peter; Kumlehn, Jochen

    2015-01-01

    Pollen embryogenesis provides a useful means of generating haploid plants for plant breeding and basic research. Although it is well-established that the efficacy of the process can be enhanced by the provision of immature pistils as a nurse tissue, the origin and compound class of the signal molecule(s) involved is still elusive. Here, a micro-culture system was established to enable the culturing of populations of barley pollen at a density too low to allow unaided embryogenesis to occur, and this was then exploited to assess the effect of using various parts of the pistil as nurse tissue. A five-fold increase in the number of embryogenic calli formed was obtained by simply cutting the pistils in half. The effectiveness of the pistil-conditioned medium was transitory, since it needed replacement at least every 4 days to measurably ensure embryogenic development. The differential effect of various size classes of compounds present in the pistil-conditioned medium showed that the relevant molecule(s) was of molecular weight below 3 kDa. This work narrows down possible feeder molecules to lower molecular weight compounds and showed that the cellular origin of the active compound(s) is not specific to any tested part of the pistil. Furthermore, the increased recovery of calli during treatment with cut pistils may provide a useful tool for plant breeders and researchers using haploid technology in barley and other plant species. PMID:26217352

  15. Cellular dynamics during early barley pollen embryogenesis revealed by time-lapse imaging

    PubMed Central

    Daghma, Diaa Eldin S.; Hensel, Goetz; Rutten, Twan; Melzer, Michael; Kumlehn, Jochen

    2014-01-01

    Plants display a remarkable capacity for cellular totipotency. An intriguing and useful example is that immature pollen cultured in vitro can pass through embryogenic development to form haploid or doubled haploid plants. However, a lack of understanding the initial mechanisms of pollen embryogenesis hampers the improvement and more effective and widespread employment of haploid technology in plant research and breeding. To investigate the cellular dynamics during the onset of pollen embryogenesis, we used time-lapse imaging along with transgenic barley expressing nuclear localized Green Fluorescent Protein. The results enabled us to identify nine distinct embryogenic and non-embryogenic types of pollen response to the culture conditions. Cell proliferation in embryogenic pollen normally started via a first symmetric mitosis (54.3% of pollen observed) and only rarely did so via asymmetric pollen mitosis I (4.3% of pollen observed). In the latter case, proliferation generally originated from the vegetative-like cell, albeit the division of the generative-like cell was observed in few types of pollen. Under the culture conditions used, fusion of cell nuclei was the only mechanism of genome duplication observed. PMID:25538715

  16. Morphometry by computerized three-dimensional reconstruction of the human carpal bones during embryogenesis.

    PubMed

    Durand, S; Delmas, V; Ho Ba Tho, M-C; Batchvarova, Z; Uhl, J F; Oberlin, C

    2006-08-01

    Carpal skeleton shows drastic developmental changes during embryogenesis. At this stage, the cartilaginous matrices appear and later form models of the limb bones. The purpose of this study was to investigate the morphometry of carpal bones in humans during embryological development. We obtained digitalized histological serial sections of 18 human embryos and early fetuses from the Institute of Anatomy in Paris. Surfdriver and MSC.Patran software were used for three-dimensional reconstruction and morphometry. There was a strong correlation between the volume of the carpal cartilaginous structure and the size of the embryos (P<0.001) and an exponential correlation between the carpal volume and the percentage of volume presented by the proximal carpal row (P=0.005). According to inertia parameters, the geometry of carpal cartilaginous structure, initially plane, becomes curved during embryogenesis. Carpal bones growth follows non-homothetic transformation. The innovations in embryo reconstruction serve as new tool for scientific investigation. A hypothesis of carpal development is proposed. PMID:16838087

  17. Widely Expressed Af17 is likely not required for Embryogenesis, Hematopoiesis, and Animal Survival

    PubMed Central

    Zhang, Zhijing; Huang, Le; Reisenauer, Mary Rose; Wu, Hongyu; Chen, Lihe; Zhang, Yujin; Xia, Yang; Zhang, Wenzheng

    2010-01-01

    As a putative transcription factor, Af17 may play a role in multiple signaling pathways. However, the Af17 expression profile during development and in adult tissues remains largely uncharacterized. The importance of Af17 function in embryogenesis, hematopoiesis and animal survival has never been addressed before. Here we report the generation of the first Af17 mutant mouse model, and characterization of the Af17 temporal and spatial expression profile in various embryonic stages and adult tissues by X-gal staining, in situ hybridization and RT-PCR. Af17 expression is detected in specific cell populations in all stages and in multiple tissues examined. In situ hybridization yielded a consistent Af17 expression pattern by X-gal staining. Homozygous mutant mice are viable, fertile, normal in size, and do not display any gross physical, behavioral or hematopoietic abnormalities. Thus, our studies describe the generation of the first Af17 mutant mouse model, provide the first developmental profile of Af17 expression, and reveal that Af17 may be dispensable for normal embryogenesis, hematopoiesis, and animal survival. PMID:21170927

  18. Embryogenesis and larval biology of the cold-water coral Lophelia pertusa.

    PubMed

    Larsson, Ann I; Järnegren, Johanna; Strömberg, Susanna M; Dahl, Mikael P; Lundälv, Tomas; Brooke, Sandra

    2014-01-01

    Cold-water coral reefs form spectacular and highly diverse ecosystems in the deep sea but little is known about reproduction, and virtually nothing about the larval biology in these corals. This study is based on data from two locations of the North East Atlantic and documents the first observations of embryogenesis and larval development in Lophelia pertusa, the most common framework-building cold-water scleractinian. Embryos developed in a more or less organized radial cleavage pattern from ∼ 160 µm large neutral or negatively buoyant eggs, to 120-270 µm long ciliated planulae. Embryogenesis was slow with cleavage occurring at intervals of 6-8 hours up to the 64-cell stage. Genetically characterized larvae were sexually derived, with maternal and paternal alleles present. Larvae were active swimmers (0.5 mm s(-1)) initially residing in the upper part of the water column, with bottom probing behavior starting 3-5 weeks after fertilization. Nematocysts had developed by day 30, coinciding with peak bottom-probing behavior, and possibly an indication that larvae are fully competent to settle at this time. Planulae survived for eight weeks under laboratory conditions, and preliminary results indicate that these planulae are planktotrophic. The late onset of competency and larval longevity suggests a high dispersal potential. Understanding larval biology and behavior is of paramount importance for biophysical modeling of larval dispersal, which forms the basis for predictions of connectivity among populations. PMID:25028936

  19. Biological and biochemical properties of two Xenopus laevis N-acetylgalactosaminyltransferases with contrasting roles in embryogenesis

    PubMed Central

    Voglmeir, Josef; Laurent, Nicolas; Flitsch, Sabine L.; Oelgeschläger, Michael; Wilson, Iain B.H.

    2015-01-01

    The biosynthesis of mucin-type O-linked glycans in animals is initiated by members of the large family of polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts), which play important roles in embryogenesis, organogenesis, adult tissue homeostasis and carcinogenesis. Until now, the mammalian forms of these enzymes have been the best characterized. However, two N-acetylgalactosaminyltransferases (xGalNAc-T6 and xGalNAc-T16) from the African clawed frog (Xenopus laevis), which are most homologous to those encoded by the human GALNT6 and GALNT16 (GALNTL1) genes, were shown to have contrasting roles in TGF-β/BMP signaling in embryogenesis. In this study we have examined these two enzymes further and show differences in their in vivo function during X. laevis embyrogenesis as evidenced by in situ hybridization and overexpression experiments. In terms of enzymatic activity, both enzymes were found to be active towards the EA2 peptide, but display differential activity towards a peptide based on the sequence of ActR-IIB, a receptor relevant to TGF-β/BMP signaling. In summary, these data demonstrate that these two enzymes from different branches of the N-acetylgalactosaminyltransferase do not only display differential substrate specificities, but also specific and distinct expression pattern and biological activities in vivo. PMID:25447273

  20. Fetal hematopoietic stem cells express MFG-E8 during mouse embryogenesis.

    PubMed

    Lee, Jaehun; Choi, Byung-il; Park, Seo Young; An, Su Yeon; Han, Jiyou; Kim, Jong-Hoon

    2015-01-01

    The milk fat globule-EGF-factor 8 protein (MFG-E8) has been identified in various tissues, where it has an important role in intercellular interactions, cellular migration, and neovascularization. Previous studies showed that MFG-E8 is expressed in different cell types under normal and pathophysiological conditions, but its expression in hematopoietic stem cells (HSCs) during hematopoiesis has not been reported. In the present study, we investigated MFG-E8 expression in multiple hematopoietic tissues at different stages of mouse embryogenesis. Using immunohistochemistry, we showed that MFG-E8 was specifically expressed in CD34(+) HSCs at all hematopoietic sites, including the yolk sac, aorta-gonad-mesonephros region, placenta and fetal liver, during embryogenesis. Fluorescence-activated cell sorting and polymerase chain reaction analyses demonstrated that CD34(+) cells, purified from the fetal liver, expressed additional HSC markers, c-Kit and Sca-1, and that these CD34(+) cells, but not CD34(-) cells, highly expressed MFG-E8. We also found that MFG-E8 was not expressed in HSCs in adult mouse bone marrow, and that its expression was confined to F4/80(+) macrophages. Together, this study demonstrates, for the first time, that MFG-8 is expressed in fetal HSC populations, and that MFG-E8 may have a role in embryonic hematopoiesis. PMID:26206421

  1. Fetal hematopoietic stem cells express MFG-E8 during mouse embryogenesis

    PubMed Central

    Lee, Jaehun; Choi, Byung-il; Park, Seo Young; An, Su Yeon; Han, Jiyou; Kim, Jong-Hoon

    2015-01-01

    The milk fat globule-EGF-factor 8 protein (MFG-E8) has been identified in various tissues, where it has an important role in intercellular interactions, cellular migration, and neovascularization. Previous studies showed that MFG-E8 is expressed in different cell types under normal and pathophysiological conditions, but its expression in hematopoietic stem cells (HSCs) during hematopoiesis has not been reported. In the present study, we investigated MFG-E8 expression in multiple hematopoietic tissues at different stages of mouse embryogenesis. Using immunohistochemistry, we showed that MFG-E8 was specifically expressed in CD34+ HSCs at all hematopoietic sites, including the yolk sac, aorta-gonad-mesonephros region, placenta and fetal liver, during embryogenesis. Fluorescence-activated cell sorting and polymerase chain reaction analyses demonstrated that CD34+ cells, purified from the fetal liver, expressed additional HSC markers, c-Kit and Sca-1, and that these CD34+ cells, but not CD34− cells, highly expressed MFG-E8. We also found that MFG-E8 was not expressed in HSCs in adult mouse bone marrow, and that its expression was confined to F4/80+ macrophages. Together, this study demonstrates, for the first time, that MFG-8 is expressed in fetal HSC populations, and that MFG-E8 may have a role in embryonic hematopoiesis. PMID:26206421

  2. 3D Light-Sheet Fluorescence Microscopy of Cranial Neurons and Vasculature during Zebrafish Embryogenesis

    PubMed Central

    Park, Ok Kyu; Kwak, Jina; Jung, Yoo Jung; Kim, Young Ho; Hong, Hyun-Seok; Hwang, Byung Joon; Kwon, Seung-Hae; Kee, Yun

    2015-01-01

    Precise 3D spatial mapping of cells and their connections within living tissues is required to fully understand developmental processes and neural activities. Zebrafish embryos are relatively small and optically transparent, making them the vertebrate model of choice for live in vivo imaging. However, embryonic brains cannot be imaged in their entirety by confocal or two-photon microscopy due to limitations in optical range and scanning speed. Here, we use light-sheet fluorescence microscopy to overcome these limitations and image the entire head of live transgenic zebrafish embryos. We simultaneously imaged cranial neurons and blood vessels during embryogenesis, generating comprehensive 3D maps that provide insight into the coordinated morphogenesis of the nervous system and vasculature during early development. In addition, blood cells circulating through the entire head, vagal and cardiac vasculature were also visualized at high resolution in a 3D movie. These data provide the foundation for the construction of a complete 4D atlas of zebrafish embryogenesis and neural activity. PMID:26429501

  3. Mutation dependance of the mitochondrial DNA copy number in the first stages of human embryogenesis.

    PubMed

    Monnot, Sophie; Samuels, David C; Hesters, Laetitia; Frydman, Nelly; Gigarel, Nadine; Burlet, Philippe; Kerbrat, Violaine; Lamazou, Frédéric; Frydman, René; Benachi, Alexandra; Feingold, Josué; Rotig, Agnes; Munnich, Arnold; Bonnefont, Jean-Paul; Steffann, Julie

    2013-05-01

    Mitochondrial DNA (mtDNA) content is thought to remain stable over the preimplantation period of human embryogenesis that is, therefore, suggested to be entirely dependent on ooplasm mtDNA capital. We have explored the impact of two disease-causing mutations [m.3243A>G myopathy, encephalopathy, lactic acidosis and stroke-like syndrome (MELAS) and m.8344A>G myoclonic epilepsy associated with ragged-red fibers (MERRF)] on mtDNA amounts in human oocytes and day 4-5 preimplantation embryos. The mtDNA amount was stable in MERRF and control materials, whereas gradually increasing from the germinal vesicle of oogenesis to the blastocyst stage of embryogenesis in MELAS cells, MELAS embryos carrying ∼3-fold higher mtDNA amount than control embryos (P = 0.0003). A correlation between mtDNA copy numbers and mutant loads was observed in MELAS embryos (R(2) = 0.42, P < 0.0013), suggestive of a compensation for the respiratory chain defect resulting from high mutation levels. These results suggest that mtDNA can replicate in early embryos and emphasize the need for sufficient amount of wild-type mtDNA to sustain embryonic development in humans. PMID:23390135

  4. Effect of microgravity and hypergravity on embryo axis alignment during postencystment embryogenesis in Artemia franciscana (Anostraca)

    NASA Technical Reports Server (NTRS)

    Rosowski, J. R.; Gouthro, M. A.; Schmidt, K. K.; Klement, B. J.; Spooner, B. S.

    1995-01-01

    Cysts of brine shrimp attached with a liquid adhesive to 12-mm diameter glass coverslips in a syringe-type fluid processing apparatus were flown aboard the NASA space shuttle Discovery, flight STS-60, from 3-11 February 1994, and were allowed to undergo postencystment embryogenesis and to hatch in microgravity. The shuttle flight and the ground-based control coverslips with attached cysts were parallel to the earth's surface during incubation in salt water. Based on the position of the cyst shell crack in the attached cyst population, the ground-control nauplii emerged mostly upward. On the shuttle in microgravity, although our method of detection of orientation would not reveal emergence toward the coverslip, the ratio of the position of the cyst shell crack in the population after hatching best fit the predicted values of a random direction for nauplii emergence. Centrifugation on earth was then used to create hypergravity forces of up to 73 g during postencystment embryogenesis and hatching. The upward orientation of emerging nauplii showed a high degree of correlation (r(2) =98.8%) with a linear relationship to the log of g, with 78.2% of the total hatching upward at 1 g and 91.0% hatching upward at 73 g.

  5. The RPN1 subunit of the 26S proteasome in Arabidopsis is essential for embryogenesis.

    PubMed

    Brukhin, Vladimir; Gheyselinck, Jacqueline; Gagliardini, Valeria; Genschik, Pascal; Grossniklaus, Ueli

    2005-10-01

    The 26S proteasome plays a central role in the degradation of regulatory proteins involved in a variety of developmental processes. It consists of two multisubunit protein complexes: the proteolytic core protease and the regulatory particle (RP). The function of most RP subunits is poorly understood. Here, we describe mutants in the Arabidopsis thaliana RPN1 subunit, which is encoded by two paralogous genes, RPN1a and RPN1b. Disruption of RPN1a caused embryo lethality, while RPN1b mutants showed no obvious abnormal phenotype. Embryos homozygous for rpn1a arrested at the globular stage with defects in the formation of the embryonic root, the protoderm, and procambium. Cyclin B1 protein was not degraded in these embryos, consistent with cell division defects. Double mutant plants (rpn1a/RPN1a rpn1b/rpn1b) produced embryos with a phenotype indistinguishable from that of the rpn1a single mutant. Thus, despite their largely overlapping expression patterns in flowers and developing seeds, the two isoforms do not share redundant functions during gametogenesis and embryogenesis. However, complementation of the rpn1a mutation with the coding region of RPN1b expressed under the control of the RPN1a promoter indicates that the two RPN1 isoforms are functionally equivalent. Overall, our data indicate that RPN1 activity is essential during embryogenesis, where it might participate in the destruction of a specific set of protein substrates. PMID:16169895

  6. The FUS3 transcription factor functions through the epidermal regulator TTG1 during embryogenesis in Arabidopsis.

    PubMed

    Tsuchiya, Yuichiro; Nambara, Eiji; Naito, Satoshi; McCourt, Peter

    2004-01-01

    Loss-of-function mutations in the FUSCA3 (FUS3) gene of Arabidopsis result in alterations in cotyledon identity, inability to complete late seed maturation processes, and the premature activation of apical and root embryonic meristems, which indicates that this transcription factor is an essential regulator of embryogenesis. Although FUS3 shows a complex pattern of expression in the embryo, this gene is only required in the protoderm to carry out its functions. Moreover, the epidermal morphogenesis regulator TRANSPARENT TESTA GLABRA1 (TTG1) is negatively regulated by FUS3 in the embryo. When a loss-of-function ttg1 mutation is introduced into a fus3 mutant, a number of fus3-related phenotypes are rescued, indicating a functional TTG1 gene is required to manifest the fus3 mutant phenotype. It therefore appears that one of the functions of FUS3 is to restrict the domain of expression of TTG1 during embryogenesis. The FUS3-TTG1 interaction is both maternal and zygotic, suggesting a complex relationship is required between these gene products to allow correct seed development. PMID:14675433

  7. 3D Light-Sheet Fluorescence Microscopy of Cranial Neurons and Vasculature during Zebrafish Embryogenesis.

    PubMed

    Park, Ok Kyu; Kwak, Jina; Jung, Yoo Jung; Kim, Young Ho; Hong, Hyun-Seok; Hwang, Byung Joon; Kwon, Seung-Hae; Kee, Yun

    2015-11-30

    Precise 3D spatial mapping of cells and their connections within living tissues is required to fully understand developmental processes and neural activities. Zebrafish embryos are relatively small and optically transparent, making them the vertebrate model of choice for live in vivo imaging. However, embryonic brains cannot be imaged in their entirety by confocal or two-photon microscopy due to limitations in optical range and scanning speed. Here, we use light-sheet fluorescence microscopy to overcome these limitations and image the entire head of live transgenic zebrafish embryos. We simultaneously imaged cranial neurons and blood vessels during embryogenesis, generating comprehensive 3D maps that provide insight into the coordinated morphogenesis of the nervous system and vasculature during early development. In addition, blood cells circulating through the entire head, vagal and cardiac vasculature were also visualized at high resolution in a 3D movie. These data provide the foundation for the construction of a complete 4D atlas of zebrafish embryogenesis and neural activity. PMID:26429501

  8. Embryogenesis and Larval Biology of the Cold-Water Coral Lophelia pertusa

    PubMed Central

    Strömberg, Susanna M.; Dahl, Mikael P.; Lundälv, Tomas; Brooke, Sandra

    2014-01-01

    Cold-water coral reefs form spectacular and highly diverse ecosystems in the deep sea but little is known about reproduction, and virtually nothing about the larval biology in these corals. This study is based on data from two locations of the North East Atlantic and documents the first observations of embryogenesis and larval development in Lophelia pertusa, the most common framework-building cold-water scleractinian. Embryos developed in a more or less organized radial cleavage pattern from ∼160 µm large neutral or negatively buoyant eggs, to 120–270 µm long ciliated planulae. Embryogenesis was slow with cleavage occurring at intervals of 6–8 hours up to the 64-cell stage. Genetically characterized larvae were sexually derived, with maternal and paternal alleles present. Larvae were active swimmers (0.5 mm s−1) initially residing in the upper part of the water column, with bottom probing behavior starting 3–5 weeks after fertilization. Nematocysts had developed by day 30, coinciding with peak bottom-probing behavior, and possibly an indication that larvae are fully competent to settle at this time. Planulae survived for eight weeks under laboratory conditions, and preliminary results indicate that these planulae are planktotrophic. The late onset of competency and larval longevity suggests a high dispersal potential. Understanding larval biology and behavior is of paramount importance for biophysical modeling of larval dispersal, which forms the basis for predictions of connectivity among populations. PMID:25028936

  9. The Maize MADS Box Gene ZmMADS3 Affects Node Number and Spikelet Development and Is Co-Expressed with ZmMADS1 during Flower Development, in Egg Cells, and Early Embryogenesis1

    PubMed Central

    Heuer, Sigrid; Hansen, Susanne; Bantin, Jrg; Brettschneider, Reinhold; Kranz, Erhard; Lrz, Horst; Dresselhaus, Thomas

    2001-01-01

    MADS box genes represent a large gene family of transcription factors with essential functions during flower development and organ differentiation processes in plants. Addressing the question of whether MADS box genes are involved in the regulation of the fertilization process and early embryo development, we have isolated two novel MADS box cDNAs, ZmMADS1 and ZmMADS3, from cDNA libraries of maize (Zea mays) pollen and egg cells, respectively. The latter gene is allelic to ZAP1. Transcripts of both genes are detectable in egg cells and in in vivo zygotes of maize. ZmMADS1 is additionally expressed in synergids and in central and antipodal cells. During early somatic embryogenesis, ZmMADS1 expression is restricted to cells with the capacity to form somatic embryos, and to globular embryos at later stages. ZmMADS3 is detectable only by more sensitive reverse transcriptase-PCR analyses, but is likewise expressed in embryogenic cultures. Both genes are not expressed in nonembryogenic suspension cultures and in isolated immature and mature zygotic embryos. During flower development, ZmMADS1 and ZmMADS3 are co-expressed in all ear spikelet organ primordia at intermediate stages. Among vegetative tissues, ZmMADS3 is expressed in stem nodes and displays a gradient with highest expression in the uppermost node. Transgenic maize plants ectopically expressing ZmMADS3 are reduced in height due to a reduced number of nodes. Reduction of seed set and male sterility were observed in the plants. The latter was due to absence of anthers. Putative functions of the genes during reproductive and vegetative developmental processes are discussed. PMID:11553732

  10. HNF4beta, a new gene of the HNF4 family with distinct activation and expression profiles in oogenesis and embryogenesis of Xenopus laevis.

    PubMed Central

    Holewa, B; Zapp, D; Drewes, T; Senkel, S; Ryffel, G U

    1997-01-01

    The transcription factor hepatocyte nuclear factor 4 (HNF4) is an orphan member of the nuclear receptor superfamily expressed in mammals in liver, kidney, and the digestive tract. Recently, we isolated the Xenopus homolog of mammalian HNF4 and revealed that it is not only a tissue-specific transcription factor but also a maternal component of the Xenopus egg and distributed within an animal-to-vegetal gradient. We speculate that this gradient cooperates with the vegetally localized embryonic induction factor activin A to activate expression of HNF1alpha, a tissue-specific transcription factor with an expression pattern overlapping that of HNF4. We have now identified a second Xenopus HNF4 gene, which is more distantly related to mammalian HNF4 than the previously isolated gene. This new gene was named HNF4beta to distinguish it from the known HNF4 gene, which is now called HNF4alpha. By reverse transcription-PCR, we detected within the 5' untranslated region of HNF4beta two splice variants (HNF4beta2 and HNF4beta3) with additional exons, which seem to affect RNA stability. HNF4beta is a functional transcription factor acting sequence specifically on HNF4 binding sites known for HNF4alpha, but it seems to have a lower DNA binding activity and is a weaker transactivator than the alpha isoform. Furthermore, the two factors differ with respect to tissue distribution in adult frogs: whereas HNF4alpha is expressed in liver and kidney, HNF4beta is expressed in addition in stomach, intestine, lung, ovary, and testis. Both factors are maternal proteins and present at constant levels throughout embryogenesis. However, using reverse transcription-PCR, we found the RNA levels to change substantially: whereas HNF4alpha is expressed early during oogenesis and is absent in the egg, HNF4beta is first detected in the latest stage of oogenesis, and transcripts are present in the egg and early cleavage stages. Furthermore, zygotic HNF4alpha transcripts appear in early gastrula and accumulate during further embryogenesis, whereas HNF4beta mRNA transiently appears during gastrulation before it accumulates again at the tail bud stage. All of these distinct characteristics of the newly identified HNF4 protein imply that the alpha and beta isoform have different functions in development and in adult tissues. PMID:9001222

  11. Assessing the New DSM-5 Diagnosis of Somatic Symptom Disorder.

    PubMed

    Barsky, Arthur J

    2016-01-01

    The conceptualization of somatization and what were previously termed somatoform disorders has changed substantially in the DSM-5 compared with previous diagnostic systems. The current diagnostic criteria for somatic symptom disorder (SSD) require the presence of symptoms (Criterion A) combined with a substantial impact of these symptoms on thoughts, emotions, and behaviors (Criterion B). In this issue of Psychosomatic Medicine, Toussaint et al. describe the development and empirical validation of a self-report questionnaire -the SSD-12- to assess the new psychological criteria (the "B criteria") of DSM-5 SSD. This is an important contribution because previously there was no questionnaire available to assess the B Criterion of SSD. The new DSM-5 criteria for SSD no longer require the absence of an adequate medical explanation for a somatic symptom, but rather define positive diagnostic criteria, focusing on the psychological impact of the somatic symptoms rather than their purported (medical) cause. Although this new conceptualization of somatization-related disorders has several advantages, seriously ill medical patients may well score high on the B Criterion for SSD on that basis alone and not because their psychological response to the medical illness is disproportionate or excessive. Measures of medical morbidity therefore need to be included in the interpretation of the SSD in individuals with severe medical conditions. Given the revised DSM-5 criteria, the newly developed and validated SSD-12 is a useful tool for diagnosing and monitoring treatment response in SSD. PMID:26599912

  12. Developmental regulation of neuroligin genes in Japanese rice fish (oryzias latipes) embryogenesis maintains the rhythym during ethanol-in

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although prenatal alcohol exposure is the potential cause of fetal alcohol spectrum disorder (FASD) in humans, the molecular mechanism(s) of FASD is yet unknown. We have used Japanese rice fish (Oryzias latipes) embryogenesis as an animal model of FASD and reported that this model has effectively ge...

  13. E3 ubiquitin ligases promote progression of differentiation during C. elegans embryogenesis.

    PubMed

    Du, Zhuo; He, Fei; Yu, Zidong; Bowerman, Bruce; Bao, Zhirong

    2015-02-15

    Regulated choice between cell fate maintenance and differentiation provides decision points in development to progress toward more restricted cell fates or to maintain the current one. Caenorhabditis elegans embryogenesis follows an invariant cell lineage where cell fate is generally more restricted upon each cell division. EMS is a progenitor cell in the four-cell embryo that gives rise to the endomesoderm. We recently found that when ubiquitin-mediated protein degradation is compromised, the anterior daughter of EMS, namely MS, reiterates the EMS fate. This observation demonstrates an essential function of ubiquitin-mediated protein degradation in driving the progression of EMS-to-MS differentiation. Here we report a genome-wide screen of the ubiquitin pathway and extensive lineage analyses. The results suggest a broad role of E3 ligases in driving differentiation progression. First, we identified three substrate-binding proteins for two Cullin-RING ubiquitin ligase (CRL) E3 complexes that promote the progression from the EMS fate to MS, namely LIN-23/β-TrCP and FBXB-3 for the CRL1/SCF complex and ZYG-11/ZYG-11B for the CRL2 complex. Genetic analyses suggest these E3 ligases function through a multifunctional protein OMA-1 and the endomesoderm lineage specifier SKN-1 to drive differentiation. Second, we found that depletion of components of the CRL1/SCF complex induces fate reiteration in all major founder cell lineages. These data suggest that regulated choice between self-renewal and differentiation is widespread during C. elegans embryogenesis as in organisms with regulative development, and ubiquitin-mediated protein degradation drives the choice towards differentiation. Finally, bioinformatic analysis of time series gene expression data showed that expression of E3 genes is transiently enriched during time windows of developmental stage transitions. Transcription factors show similar enrichment, but not other classes of regulatory genes. Based on these findings we propose that ubiquitin-mediated protein degradation, like many transcription factors, function broadly as regulators driving developmental progression during embryogenesis in C. elegans. PMID:25523393

  14. Molecular characterization of zebrafish embryogenesis via DNA microarrays and multiplatform time course metabolomics studies.

    PubMed

    Soanes, Kelly H; Achenbach, John C; Burton, Ian W; Hui, Joseph P M; Penny, Susanne L; Karakach, Tobias K

    2011-11-01

    One of the greatest strengths of "-omics" technologies is their ability to capture a molecular snapshot of multiple cellular processes simultaneously. Transcriptomics, proteomics, and metabolomics have, individually, been used in wide-ranging studies involving cell lines, tissues, model organisms, and human subjects. Nonetheless, despite the fact that their power lies in the global acquisition of parallel data streams, these methods continue to be employed separately. We highlight work done to merge transcriptomics and metabolomics technologies to study zebrafish (Danio rerio) embryogenesis. We combine information from three bioanalytical platforms, that is, DNA microarrays, (1)H nuclear magnetic resonance ((1)H NMR), and mass spectrometry (MS)-based metabolomics, to identify and provide insights into the organism's developmental regulators. We apply a customized approach to the analysis of such time-ordered measurements to provide temporal profiles that depict the modulation of metabolites and gene transcription. Initially, the three data sets were analyzed individually but later they were fused to highlight the advantages gained through such an integrated approach. Unique challenges posed by fusion of such data are discussed given differences in the measurement error structures, the wide dynamic range for the molecular species, and the analytical platforms used to measure them (i.e., fluorescence ratios, NMR, and MS intensities). Our data analysis reveals that changes in transcript levels at specific developmental stages correlate with previously published data with over 90% accuracy. In addition, transcript profiles exhibited trends that were similar to the accumulation of metabolites over time. Profiles for metabolites such as choline-like compounds (Trimethylamine-N-oxide, phosphocholine, betaine), creatinine/creatine, and other metabolites involved in energy metabolism exhibited a steady increase from 15 hours post fertilization (hpf) to 48 hpf. Other metabolite and transcript profiles were transiently rising and then falling back to baseline. The "house keeping" metabolites such as branched chain amino acids exhibited a steady presence throughout embryogenesis. Although the transcript profiling corresponds to only 16 384 genes, a subset of the total number of genes in the zebrafish genome, we identified examples where gene transcript and metabolite profiles correlate with one another, reflective of a relationship between gene and metabolite regulation over the course of embryogenesis. PMID:21910437

  15. E3 ubiquitin ligases promote progression of differentiation during C. elegans embryogenesis

    PubMed Central

    Du, Zhuo; He, Fei; Yu, Zidong; Bowerman, Bruce; Bao, Zhirong

    2014-01-01

    Regulated choice between cell fate maintenance and differentiation provides decision points in development to progress toward more restricted cell fates or to maintain the current one. C. elegans embryogenesis follows an invariant cell lineage where cell fate is generally more restricted upon each cell division. EMS is a progenitor cell in the four-cell embryo that gives rise to the endomesoderm. We recently found that when ubiquitin-mediated protein degradation is compromised, the anterior daughter of EMS, namely MS, reiterates the EMS fate. This observation demonstrates an essential function of ubiquitin-mediated protein degradation in driving the progression of EMS-to-MS differentiation. Here we report a genome-wide screen of the ubiquitin pathway and extensive lineage analyses. The results suggest a broad role of E3 ligases in driving differentiation progression. First, we identified three substrate-binding proteins for two CRL (Cullin-RING ubiquitin Ligase) E3 complexes that promote the progression from the EMS fate to MS, namely LIN-23/β-TrCP and FBXB-3 for the CRL1/SCF complex and ZYG-11/ZYG-11B for the CRL2 complex. Genetic analyses suggest these E3 ligases function through a multifunctional protein OMA-1 and the endomesoderm lineage specifier SKN-1 to drive differentiation. Second, we found that depletion of components of the CRL1/SCF complex induces fate reiteration in all major founder cell lineages. These data suggest that regulated choice between self-renewal and differentiation is widespread during C. elegans embryogenesis as in organisms with regulative development, and ubiquitin-mediated protein degradation drives the choice towards differentiation. Finally, bioinformatic analysis of time series gene expression data showed that expression of E3 genes is transiently enriched during time windows of developmental stage transitions. Transcription factors show similar enrichment, but not other classes of regulatory genes. Based on these findings we propose that ubiquitin-mediated protein degradation, like many transcription factors, function broadly as regulators driving developmental progression during embryogenesis in C. elegans. PMID:25523393

  16. Chick embryogenesis: a unique platform to study the effects of environmental factors on embryo development.

    PubMed

    Yahav, S; Brake, J

    2014-01-01

    Bird embryogenesis takes place in a relatively protected environment that can be manipulated especially well in domestic fowl (chickens) where incubation has long been a commercial process. The embryonic developmental process has been shown to begin in the oviduct such that the embryo has attained either the blastodermal and/or gastrulation stage of development at oviposition. Bird embryos can be affected by "maternal effects," and by environmental conditions during the pre-incubation and incubation periods. "Maternal effects" has been described as an evolutionary mechanism that has provided the mother, by hormonal deposition into the yolk, with the potential to proactively influence the development of her progeny by exposing them to her particular hormonal pattern in such a manner as to influence their ability to cope with the expected wide range of environmental conditions that may occur post-hatching. Another important aspect of "maternal effects" is the effect of the maternal nutrient intake on progeny traits. From a commercial broiler chicken production perspective, it has been established that greater cumulative nutrient intake by the hen during her pullet rearing phase prior to photostimulation resulted in faster growing broiler progeny. Generally, maternal effects on progeny, which have both a genetic and an environmental component represented by yolk hormones deposition and embryo nutrient utilization, have an important effect on the development of a wide range of progeny traits. Furthermore, commercial embryo development during pre-incubation storage and incubation, as well as during incubation per se has been shown to largely depend upon temperature, while other environmental factors that include egg position during storage, and the amount of H2O and CO2 lost by the egg and the subsequent effect on albumen pH and height during storage have become important environmental factors to be considered for successful embryogenesis under commercial conditions. Manipulating environmental temperature during the period of egg storage, during the intermediate pre-incubation period, and incubation period per se has been found to significantly affect embryo development, hatching progress, chick quality at hatching, and chick development post-hatching. These temperature manipulations have also been shown to affect the acquisition of thermotolerance to subsequent post-hatching thermal challenge. This chapter will focus on: a. "maternal effects" on embryo and post-hatching development; b. environmental effects during the post-ovipositional period of egg storage, the intermediate pre-incubation period, and incubation period per se on chick embryogenesis and subsequent post-hatching growth and development; and c. effects of temperature manipulations during the pre-incubation and incubation periods on acquisition of thermotolerance and development of secondary sexual characteristics in broiler chickens. PMID:25158087

  17. Somatic cells count in cow's bulk tank milk.

    PubMed

    Olechnowicz, Jan; Ja?kowski, Jedrzej M

    2012-06-01

    The objective of this study was therefore to present factors affecting somatic cell counts in bovine bulk milk as a result of intramammary infections as well as non-infectious factors. The paper presents also the impact of on-farm management practices on the level of bulk milk somatic cell counts and presents quality indicators in bulk tank milk. At the farm level bulk milk bacterial infection takes place through three main sources: bacterial contamination from the external surface of the udder and teats, from the surface of the milking equipment, and from mastitis microorganisms within the udder. The threshold of 200,000 cells/ml identifies bacteriological negative quarters of the udder. The counts of mammary pathogens in bulk tank milk are relatively low, on average not exceeding 1,000 cfu/ml. Environmental pathogens predominate in bulk tank milk samples with somatic cells count <300 10(3) ml. PMID:22230979

  18. Response to Propranolol and Diazepam in Somatic and Psychic Anxiety

    PubMed Central

    Tyrer, P. J.; Lader, M. H.

    1974-01-01

    A total of 12 chronically anxious psychiatric outpatients were treated with racemic propranolol (Inderal), diazepam (Valium), and placebo for one week each, using a balanced cross-over experimental design. Six patients had predominantly somatic anxiety, complaining mostly of bodily symptoms, and six had mainly psychic anxiety, complaining primarily of psychological symptoms. Clinical ratings of anxiety were made by patient and psychiatrist after each treatment. Though diazepam was in general more effective than propranolol or placebo in relieving anxiety, propranolol was more effective than placebo in patients with somatic anxiety but not in those with psychic anxiety. We suggest that propranolol should be reserved for patients whose anxiety symptoms are mainly somatic. PMID:4595181

  19. Mind-Body Interactions in Anxiety and Somatic Symptoms.

    PubMed

    Mallorquí-Bagué, Núria; Bulbena, Antonio; Pailhez, Guillem; Garfinkel, Sarah N; Critchley, Hugo D

    2016-01-01

    Anxiety and somatic symptoms have a high prevalence in the general population. A mechanistic understanding of how different factors contribute to the development and maintenance of these symptoms, which are highly associated with anxiety disorders, is crucial to optimize treatments. In this article, we review recent literature on this topic and present a redefined model of mind-body interaction in anxi