Science.gov

Sample records for somatic embryogenesis induced

  1. Somatic embryogenesis - Stress-induced remodeling of plant cell fate.

    PubMed

    Fehér, Attila

    2015-04-01

    Plants as sessile organisms have remarkable developmental plasticity ensuring heir continuous adaptation to the environment. An extreme example is somatic embryogenesis, the initiation of autonomous embryo development in somatic cells in response to exogenous and/or endogenous signals. In this review I briefly overview the various pathways that can lead to embryo development in plants in addition to the fertilization of the egg cell and highlight the importance of the interaction of stress- and hormone-regulated pathways during the induction of somatic embryogenesis. Somatic embryogenesis can be initiated in planta or in vitro, directly or indirectly, and the requirement for dedifferentiation as well as the way to achieve developmental totipotency in the various systems is discussed in light of our present knowledge. The initiation of all forms of the stress/hormone-induced in vitro as well as the genetically provoked in planta somatic embryogenesis requires extensive and coordinated genetic reprogramming that has to take place at the chromatin level, as the embryogenic program is under strong epigenetic repression in vegetative plant cells. Our present knowledge on chromatin-based mechanisms potentially involved in the somatic-to-embryogenic developmental transition is summarized emphasizing the potential role of the chromatin to integrate stress, hormonal, and developmental pathways leading to the activation of the embryogenic program. The role of stress-related chromatin reorganization in the genetic instability of in vitro cultures is also discussed. This article is part of a Special Issue entitled: Stress as a fundamental theme in cell plasticity.

  2. Involvement of polyamine biosynthesis in somatic embryogenesis of Valencia sweet orange (Citrus sinensis) induced by glycerol.

    PubMed

    Wu, Xiao-Ba; Wang, Jing; Liu, Ji-Hong; Deng, Xiu-Xin

    2009-01-01

    Culture of Citrus sinensis embryogenic callus on the embryo-inducing medium (EIM) containing glycerol gave rise to a large number of embryos, whereas very few embryos were observed on the callus growth medium (CGM). In the current paper, attempts were made to investigate whether polyamine biosynthesis was involved in glycerol-mediated somatic embryogenesis. Quantification of free polyamines by high-performance liquid chromatography showed that the cultures on EIM had less putrescine than those on CGM. However, increase in spermidine and spermine was detected in cultures on EIM during the first 20d of culture, coincident with abundant somatic embryogenesis. The globular embryos contained more polyamines than embryos at other stages. Semi-quantitative reverse transcriptase-polymerase chain reaction assay showed that expression levels of all of the five key genes involved in polyamine biosynthesis, with the exception of S-adenosylmethionine decarboxylase, were induced in cultures on EIM, and that their transcriptional levels were increased with maturation of the embryos. Addition of alpha-difluoromethylornithine, a polyamine biosynthesis inhibitor, to EIM resulted in remarkable inhibition of somatic embryogenesis, concurrent with notable reduction of endogenous putrescine and spermidine, particularly at higher concentrations. Exogenous application of 1mM putrescine to EIM together with 5mM alpha-difluoromethylornithine led to dramatic enhancement of endogenous polyamines, which successfully restored somatic embryogenesis. All of these, collectively, demonstrated that free polyamines, at least spermidine and spermine herein, were involved in glycerol-mediated promotion of somatic embryogenesis, which will open a new avenue for establishing a sophisticated system for somatic embryogenesis based on the modulation of endogenous polyamines.

  3. Cytokinin induced somatic embryogenesis from immature embryos of Abies nordmanniana Lk.

    PubMed

    Viktor Nørgaard, J; Krogstrup, P

    1991-01-01

    Abies nordmanniana Lk. is used in short intensive rotations for Christmas tree production. Thus there is a high demand for development of advanced propagation and breeding methods. Somatic embryogenesis was easily induced from immature (precotyledonary) embryos collected in July 1989 with cytokinin as the sole plant growth regulator. The proliferating embryogenic cell masses were characteristic of conifer somatic embryogenesis and could be maintained on a simple basal medium containing 5 μM benzylaminopurine. Auxin inhibited induction as well as proliferation. Proliferation was improved by up to 30 % by addition of L-glutamine and/or casein hydrolysate. Neither cytokinin concentration nor culture on 3 different basal media, differing markedly in their nitrogen composition, affected the proliferation rate. Embryos matured using a 4 week subculture on medium containing 10 μM abscisic acid and subsequent transfer to medium devoid of plant growth regulators.

  4. Application of Somatic Embryogenesis in Woody Plants.

    PubMed Central

    Guan, Yuan; Li, Shui-Gen; Fan, Xiao-Fen; Su, Zhen-Hong

    2016-01-01

    Somatic embryogenesis is a developmental process where a plant somatic cell can dedifferentiate to a totipotent embryonic stem cell that has the ability to give rise to an embryo under appropriate conditions. This new embryo can further develop into a whole plant. In woody plants, somatic embryogenesis plays a critical role in clonal propagation and is a powerful tool for synthetic seed production, germplasm conservation, and cryopreservation. A key step in somatic embryogenesis is the transition of cell fate from a somatic cell to embryo cell. Although somatic embryogenesis has already been widely used in a number of woody species, propagating adult woody plants remains difficult. In this review, we focus on molecular mechanisms of somatic embryogenesis and its practical applications in economic woody plants. Furthermore, we propose a strategy to improve the process of somatic embryogenesis using molecular means. PMID:27446166

  5. Thidiazuron induces shoot organogenesis at low concentrations and somatic embryogenesis at high concentrations on leaf and petiole explants of African violet (Saintpaulia ionantha Wendl).

    PubMed

    Mithila, J; Hall, J C; Victor, J M R; Saxena, P K

    2003-01-01

    Regeneration via shoot organogenesis and somatic embryogenesis was observed from thidiazuron (TDZ)-treated leaf and petiole explants of greenhouse- and in vitro-grown African violet plants. The response of cultures to other growth regulators over a range of 0.5 microM to 10 microM was 50% less than that observed with TDZ. A comparative study among several cultivars of African violet indicated that "Benjamin" and "William" had the highest regeneration potential. In "Benjamin", higher frequencies of shoot organogenesis (twofold) and somatic embryogenesis (a 50% increase) were observed from in vitro- and greenhouse-grown plants, respectively. At concentrations lower than 2.5 microM, TDZ induced shoot organogenesis, whereas at higher doses (5-10 microM) somatic embryos were formed. These findings provide the first report of simultaneous shoot organogenesis and somatic embryogenesis of African violet explants in response to TDZ. PMID:12789442

  6. Somatic Versus Zygotic Embryogenesis: Learning from Seeds.

    PubMed

    Winkelmann, Traud

    2016-01-01

    Plant embryogenesis is a fascinating developmental program that is very successfully established in nature in seeds. In case of in vitro somatic embryogenesis this process is subjected to several limitations such as asynchronous differentiation and further development of somatic embryos, malformations and disturbed polarity, precocious germination, lack of maturity, early loss of embryogenic potential, and strong genotypic differences in the regeneration efficiency. Several studies have shown the similarity of somatic and zygotic embryos in terms of morphological, histological, biochemical, and physiological aspects. However, pronounced differences have also been reported and refer to much higher stress levels, less accumulation of storage compounds and a missing distinction of differentiation and germination by a quiescent phase in somatic embryos. Here, an overview on recent literature describing both embryogenesis pathways, comparing somatic and zygotic embryos and analyzing the role of the endosperm is presented. By taking zygotic embryos as the reference and learning from the situation in seeds, somatic embryogenesis can be improved and optimized in order to make use of the enormous potential this regeneration pathway offers for plant propagation and breeding.

  7. Somatic Versus Zygotic Embryogenesis: Learning from Seeds.

    PubMed

    Winkelmann, Traud

    2016-01-01

    Plant embryogenesis is a fascinating developmental program that is very successfully established in nature in seeds. In case of in vitro somatic embryogenesis this process is subjected to several limitations such as asynchronous differentiation and further development of somatic embryos, malformations and disturbed polarity, precocious germination, lack of maturity, early loss of embryogenic potential, and strong genotypic differences in the regeneration efficiency. Several studies have shown the similarity of somatic and zygotic embryos in terms of morphological, histological, biochemical, and physiological aspects. However, pronounced differences have also been reported and refer to much higher stress levels, less accumulation of storage compounds and a missing distinction of differentiation and germination by a quiescent phase in somatic embryos. Here, an overview on recent literature describing both embryogenesis pathways, comparing somatic and zygotic embryos and analyzing the role of the endosperm is presented. By taking zygotic embryos as the reference and learning from the situation in seeds, somatic embryogenesis can be improved and optimized in order to make use of the enormous potential this regeneration pathway offers for plant propagation and breeding. PMID:26619857

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. Hemoglobins, programmed cell death and somatic embryogenesis.

    PubMed

    Hill, Robert D; Huang, Shuanglong; Stasolla, Claudio

    2013-10-01

    Programmed cell death (PCD) is a universal process in all multicellular organisms. It is a critical component in a diverse number of processes ranging from growth and differentiation to response to stress. Somatic embryogenesis is one such process where PCD is significantly involved. Nitric oxide is increasingly being recognized as playing a significant role in regulating PCD in both mammalian and plant systems. Plant hemoglobins scavenge NO, and evidence is accumulating that events that modify NO levels in plants also affect hemoglobin expression. Here, we review the process of PCD, describing the involvement of NO and plant hemoglobins in the process. NO is an effector of cell death in both plants and vertebrates, triggering the cascade of events leading to targeted cell death that is a part of an organism's response to stress or to tissue differentiation and development. Expression of specific hemoglobins can alter this response in plants by scavenging the NO, thus, interrupting the death process. Somatic embryogenesis is used as a model system to demonstrate how cell-specific expression of different classes of hemoglobins can alter the embryogenic process, affecting hormone synthesis, cell metabolite levels and genes associated with PCD and embryogenic competence. We propose that plant hemoglobins influence somatic embryogenesis and PCD through cell-specific expression of a distinct plant hemoglobin. It is based on the premise that both embryogenic competence and PCD are strongly influenced by cellular NO levels. Increases in cellular NO levels result in elevated Zn(2+) and reactive-oxygen species associated with PCD, but they also result in decreased expression of MYC2, a transcription factor that is a negative effector of indoleacetic acid synthesis, a hormone that positively influences embryogenic competence. Cell-specific hemoglobin expression reduces NO levels as a result of NO scavenging, resulting in cell survival.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. Somatic embryogenesis for efficient micropropagation of guava (Psidium guajava L.).

    PubMed

    Akhtar, Nasim

    2013-01-01

    Guava (Psidium guajava L.) is well known for edible fruit, environment friendly pharmaceutical and commercial products for both national and international market. The conventional propagation and in vitro organogenesis do not meet the demand for the good quality planting materials. Somatic embryogenesis for efficient micropropagation of guava (P. guajava L.) has been developed to fill up the gap. Somatic embryogenesis and plantlets regeneration are achieved from 10-week post-anthesis zygotic embryo explants by 8-day inductive treatment with different concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D) on MS agar medium containing 5% sucrose. Subsequent development and maturation of somatic embryos occur after 8 days on MS basal medium supplemented with 5% sucrose without plant growth regulator. The process of somatic embryogenesis shows the highest relative efficiency in 8-day treatment of zygotic embryo explants with 1.0 mg L(-1) 2,4-D. High efficiency germination of somatic embryos and plantlet regeneration takes place on half strength semisolid MS medium amended with 3% sucrose within 2 weeks of subculture. Somatic plantlets are grown for additional 2 weeks by subculturing in MS liquid growth medium containing 3% sucrose. Well-grown plantlets from liquid medium have survived very well following 2-4 week hardening process. The protocol of somatic embryogenesis is optimized for high efficiency micropropagation of guava species.

  3. 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

  4. Somatic embryogenesis in Picea suspension cultures.

    PubMed

    Stasolla, Claudio

    2006-01-01

    Generation of somatic embryos in spruce is achieved through the execution of five steps designated as: (1) induction of embryogenic tissue, (2) maintenance of embryogenic tissue, (3) embryo development, (4) embryo maturation, and (5) conversion into plants. Depending on species and genotypes within the same species, each step must be optimized for obtaining maximum results. In general, embryogenic tissue is generated from immature and mature zygotic embryos and maintained in either liquid or solid conditions in the presence of plant growth regulators auxin and cytokinin. Initiation of embryo development in suspension cultured is induced by removal of plant growth regulators, whereas continuation of development and completion of maturation require applications of abscisic acid and imposition of a desiccation period. Both treatments are needed for conferring morphological and physiological maturation to the embryos. Mature somatic embryos are germinated in the absence of plant regulators and embryo conversion (i.e., formation of a functional shoot and root, occurs after a few weeks in culture). PMID:16673908

  5. 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

  6. 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.

  7. Clonal propagation of Cyclamen persicum via somatic embryogenesis.

    PubMed

    Winkelmann, Traud

    2010-01-01

    Cyclamen (Cyclamen persicum) is an economically important ornamental pot plant with local use as cut flower as well. Traditionally, it is propagated via seeds, but interest is given in vegetative propagation of parental lines as well as superior single plants. Somatic embryogenesis is an efficient in vitro propagation method for many cyclamen cultivars. Starting from ovules of unpollinated flowers, callus is induced and propagated in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-(gamma,gamma-dimethylallylamino)purine (2iP). Transfer to hormone-free medium results in the differentiation of somatic embryos, which afterwards germinate on the same medium. These first culture stages take about 6-7 months and are carried out in complete darkness. Two to four months after the transfer to light, plantlets develop which can be acclimatized in the greenhouse. The regenerated plants are characterized by low percentages of somaclonal variation. This protocol has proven useful not only for clonal propagation, but also for artificial seed preparation, cryopreservation, genetic transformation and protoplast regeneration.

  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'.

  9. 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

  10. 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

  11. 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.

  12. 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

  13. Advances in Conifer Somatic Embryogenesis Since Year 2000.

    PubMed

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

    2016-01-01

    This review compiles research results published over the last 14 years on conifer somatic embryogenesis (SE). Emphasis is placed on the newest findings that affect the response of seed embryos (typical explants) and shoot primordia (rare explants) to the induction of SE and long-term culture of early somatic embryos. Much research in recent years has focused on maturation of somatic embryos, with respect to both yield and quality, as an important stage for the production of a large number of vigorous somatic seedlings. Attempts to scale up somatic embryo production numbers and handling have resulted in a few bioreactor designs, the utility of which may prove beneficial for an industrial application. A few simplified cryopreservation methods for embryonal masses (EM) were developed as a means to ensure cost-efficient long-term storage of genotypes during clonal field testing. Finally, recent long-term studies on the growth of somatic trees in the field, including seed production yield and comparison of seed parameters produced by somatic versus seed-derived trees, are described.

  14. 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.

  15. 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

  16. 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

  17. 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

  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. 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.

  20. 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

  1. Regulation of organogenesis and somatic embryogenesis by auxin in melon, Cucumis melo L.

    PubMed

    Tabei, Y; Kanno, T; Nishio, T

    1991-08-01

    Various tissues of seeds and seedlings of melon were cultured in vitro to study the effects of auxin concentration on organogenesis and embryogenesis. Adventitious shoots and somatic embryos were formed from explants of cotyledons of mature seeds, hypocotyls of seedlings, and leaves and petioles of young plantlets. Expanded cotyledons of seedlings formed only adventitious shoots. All tissues responded similarly to the 2,4-D concentration in the media, that is, adventitious shoots were formed at low concentration, callus proliferated without differentiation at intermediate concentration and somatic embryos were induced at high concentration. Cotyledons of mature seeds formed both adventitious shoots and somatic embryos more efficiently than any other tissues cultured.Effects of three auxins, 2,4-D, NAA and IAA, on organogenesis and embryogenesis were compared using cotyledons of mature seeds. Adventitious shoots were formed at low level of auxins (0 to 0.01 mg/l 2,4-D; 0 to 0.1 mg/l NAA; 0 to 1.0 mg/l IAA), and embryos were formed at high level of auxins (1.0 to 2.0 mg/l 2,4-D; 3.0 to 10.0 mg/l NAA; 20.0 to 100.0 mg/l IAA). IAA gave more efficient shoot formation and embryogenesis than the other auxins. PMID:24221584

  2. DNA methylation during sexual embryogenesis and implications on the induction of somatic embryogenesis in Castanea sativa Miller.

    PubMed

    Viejo, M; Rodríguez, R; Valledor, L; Pérez, M; Cañal, M J; Hasbún, R

    2010-12-01

    From anthesis to mature seed formation, burrs from cross-pollinated adult Castanea sativa Miller trees were characterized and seven developmental stages defined based on macro and micromorphological traits. In order to get an insight into the involvement of epigenetic mechanisms in sexual embryogenesis and to define somatic embryogenesis induction capability, global DNA methylation and the somatic embryogenic competence were quantified. On cross-pollinated trees once fertilization takes place, at least one ovule per ovary becomes dominant, and transient DNA demethylation occurs coinciding with the start of the sexual embryogenic programme. Unfertilized ovules from the same cluster, which maintain their prior size, increase their methylation level and undergo degeneration. These results were validated using non-cross-pollinated trees and the asynchrony of flower receptivity. When testing in vitro somatic embryogenesis response of isolated dominant ovules and axes from zygotic embryos under cross-pollinated conditions, the highest competence was found for reaching seed maturity. Thus, a "developmental window" of somatic embryogenesis in chestnut has been characterized. It includes from fertilization to embryo maturity, and a transient decrease in methylation is necessary after fertilization for the development of the somatic embryogenesis response.

  3. 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.

  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. Pine somatic embryogenesis using zygotic embryos as explants.

    PubMed

    Pullman, Gerald S; Bucalo, Kylie

    2011-01-01

    Somatic embryogenesis (SE) has the potential to be the lowest-cost method to rapidly produce large numbers of high-value somatic seedlings with desired characteristics for plantation forestry. At least 24 of the 115-120 known Pinus species can undergo SE. Initiation for most species works best with immature megagametophytes as starting material, although a few pines can initiate SE cultures from isolated mature seed embryos. Successful initiation depends heavily on explant type, embryo developmental stage, and medium salt base. Most first reports of initiation used 2,4-D and BAP or a combination of cytokinins. More recent reports have optimized initiation for many Pinus spp., but still use mostly the combinations of auxin and cytokinins. Initiation can be stimulated with medium supplements including abscisic acid (ABA), brassinosteroids, ethylene inhibitors, gibberellin inhibitors, organic acids, putrescine, specific sugar types (maltose, galactose, D-chiro-inositol, and D-xylose), triacontanol, vitamins (B12, biotin, vitamin E, and folic acid), or manipulation of environmental factors including pH, water potential, cone cold storage, gelling agent concentration, and liquid medium. Embryo development and maturation usually occur best on medium containing ABA along with water potential reduction (with sugars and polyethylene glycol) or water availability reduction (with raised gelling agent increasing gel-strength). Activated carbon and maltose may also improve embryo maturation. The main issues holding SE technology back are related to the high cost of producing a somatic seedling, incurred from low initiation percentages for recalcitrant species, culture loss, and decline after initiation and poor embryo maturation resulting in no or poor germination. Although vast progress has been made in pine SE technology over the past 24 years, fundamental studies on seed and embryo physiology, biochemistry, and gene expression are still needed to help improve the technology

  6. Tobacco arabinogalactan protein NtEPc can promote banana (Musa AAA) somatic embryogenesis.

    PubMed

    Shu, H; Xu, L; Li, Z; Li, J; Jin, Z; Chang, S

    2014-12-01

    Banana is an important tropical fruit worldwide. Parthenocarpy and female sterility made it impossible to improve banana varieties through common hybridization. Genetic transformation for banana improvement is imperative. But the low rate that banana embryogenic callus was induced made the transformation cannot be performed in many laboratories. Finding ways to promote banana somatic embryogenesis is critical for banana genetic transformation. After tobacco arabinogalactan protein gene NtEPc was transformed into Escherichia coli (DE3), the recombinant protein was purified and filter-sterilized. A series of the sterilized protein was added into tissue culture medium. It was found that the number of banana immature male flowers developing embryogenic calli increased significantly in the presence of NtEPc protein compared with the effect of the control medium. Among the treatments, explants cultured on medium containing 10 mg/l of NtEPc protein had the highest chance to develop embryogenic calli. The percentage of lines that developed embryogenic calli on this medium was about 12.5 %. These demonstrated that NtEPc protein can be used to promote banana embryogenesis. This is the first paper that reported that foreign arabinogalactan protein (AGP) could be used to improve banana somatic embryogenesis. PMID:25227688

  7. Tobacco arabinogalactan protein NtEPc can promote banana (Musa AAA) somatic embryogenesis.

    PubMed

    Shu, H; Xu, L; Li, Z; Li, J; Jin, Z; Chang, S

    2014-12-01

    Banana is an important tropical fruit worldwide. Parthenocarpy and female sterility made it impossible to improve banana varieties through common hybridization. Genetic transformation for banana improvement is imperative. But the low rate that banana embryogenic callus was induced made the transformation cannot be performed in many laboratories. Finding ways to promote banana somatic embryogenesis is critical for banana genetic transformation. After tobacco arabinogalactan protein gene NtEPc was transformed into Escherichia coli (DE3), the recombinant protein was purified and filter-sterilized. A series of the sterilized protein was added into tissue culture medium. It was found that the number of banana immature male flowers developing embryogenic calli increased significantly in the presence of NtEPc protein compared with the effect of the control medium. Among the treatments, explants cultured on medium containing 10 mg/l of NtEPc protein had the highest chance to develop embryogenic calli. The percentage of lines that developed embryogenic calli on this medium was about 12.5 %. These demonstrated that NtEPc protein can be used to promote banana embryogenesis. This is the first paper that reported that foreign arabinogalactan protein (AGP) could be used to improve banana somatic embryogenesis.

  8. 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.

  9. 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

  10. 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

  11. Influence of abscisic acid and sucrose on somatic embryogenesis in Cactus Copiapoa tenuissima Ritt. forma mostruosa.

    PubMed

    Lema-Rumińska, J; Goncerzewicz, K; Gabriel, M

    2013-01-01

    Having produced the embryos of cactus Copiapoa tenuissima Ritt. forma monstruosa at the globular stage and callus, we investigated the effect of abscisic acid (ABA) in the following concentrations: 0, 0.1, 1, 10, and 100  μ M on successive stages of direct (DSE) and indirect somatic embryogenesis (ISE). In the indirect somatic embryogenesis process we also investigated a combined effect of ABA (0, 0.1, 1  μ M) and sucrose (1, 3, 5%). The results showed that a low concentration of ABA (0-1  μ M) stimulates the elongation of embryos at the globular stage and the number of correct embryos in direct somatic embryogenesis, while a high ABA concentration (10-100  μ M) results in growth inhibition and turgor pressure loss of somatic embryos. The indirect somatic embryogenesis study in this cactus suggests that lower ABA concentrations enhance the increase in calli fresh weight, while a high concentration of 10  μ M ABA or more changes calli color and decreases its proliferation rate. However, in the case of indirect somatic embryogenesis, ABA had no effect on the number of somatic embryos and their maturation. Nevertheless, we found a positive effect of sucrose concentration for both the number of somatic embryos and the increase in calli fresh weight.

  12. Influence of Abscisic Acid and Sucrose on Somatic Embryogenesis in Cactus Copiapoa tenuissima Ritt. forma mostruosa

    PubMed Central

    Lema-Rumińska, J.; Goncerzewicz, K.; Gabriel, M.

    2013-01-01

    Having produced the embryos of cactus Copiapoa tenuissima Ritt. forma monstruosa at the globular stage and callus, we investigated the effect of abscisic acid (ABA) in the following concentrations: 0, 0.1, 1, 10, and 100 μM on successive stages of direct (DSE) and indirect somatic embryogenesis (ISE). In the indirect somatic embryogenesis process we also investigated a combined effect of ABA (0, 0.1, 1 μM) and sucrose (1, 3, 5%). The results showed that a low concentration of ABA (0-1 μM) stimulates the elongation of embryos at the globular stage and the number of correct embryos in direct somatic embryogenesis, while a high ABA concentration (10–100 μM) results in growth inhibition and turgor pressure loss of somatic embryos. The indirect somatic embryogenesis study in this cactus suggests that lower ABA concentrations enhance the increase in calli fresh weight, while a high concentration of 10 μM ABA or more changes calli color and decreases its proliferation rate. However, in the case of indirect somatic embryogenesis, ABA had no effect on the number of somatic embryos and their maturation. Nevertheless, we found a positive effect of sucrose concentration for both the number of somatic embryos and the increase in calli fresh weight. PMID:23843737

  13. Temporal Regulation of Somatic Embryogenesis by Adjusting Cellular Polyamine Content in Eggplant1

    PubMed Central

    Singh Yadav, Jitender; Venkat Rajam, Manchikatla

    1998-01-01

    Four critical stages of embryogenesis, including callus induction, cellular acquisition of morphogenetic competence, expression of embryogenic program, and development and maturation of somatic embryos during somatic embryogenesis from leaf discs of eggplant (Solanum melongena L.), were identified by scanning electron microscopy. Temporal changes in arginine decarboxylase (ADC) activity and polyamines (PAs) during critical stages of embryogenesis revealed that high levels of PAs (especially putrescine [PUT]), due to higher ADC activity in discs from the apical region (with high embryogenic capacity) than from the basal region of the leaf (with poor embryogenic capacity), were correlated with differential embryogenesis response. Kinetic studies of the up- and down-regulation of embryogenesis revealed that PUT and difluoromethylarginine pretreatments were most effective before the onset of embryogenesis. Basal discs pretreated with PUT for 4 to 7 d showed improved embryogenesis that was comparable to apical discs. PA content at various critical steps in embryogenesis from basal discs were found to be comparable to that of apical discs following adjustments of cellular PA content by PUT. In contrast, pretreatment of apical discs with difluoromethylarginine for 3 d significantly reduced ADC activity, cellular PA content, and embryogenesis to levels that were comparable to basal discs. Discs from the basal region of leaves treated with PUT for 3 d during the identified stages of embryogenesis improved their embryogenic potential. PMID:9490762

  14. Micropropagation of Codiaeum variegatum (L.) Blume and regeneration induction via adventitious buds and somatic embryogenesis.

    PubMed

    Radice, Silvia

    2010-01-01

    Codiaeum variegatum (L) Blume cv. "Corazon de oro" and cv. "Norma" are successfully micropropagated when culture are initiated with explants taken from newly sprouted shoots. The establishment and multiplication steps are possible when 1 mg/L BA or 1 mg/L IAA and 3 mg/L 2iP are added to MS medium, according to the cultivar respectively selected.Adventive organogenesis and somatic embryogenesis are induced from leaf explants taken from in vitro buds of croton. On leaf-sectioned of "Corazon de oro" cultured in vitro, 1 mg/L BA stimulates continuous somatic embryos development and induces some shoots too. Replacing BA with 1 mg/L TDZ induces up to 100% bud regeneration in the same explants. On the other hand, leaf-sectioned of C. variegatum cv. Norma does not start somatic embryo differentiation if 1 mg/L TDZ is not added to the MS basal medium. Incipient callus is observed after 30 days of culture, and then, subculture to MS with 1 mg/L BA allows the same process to show on the "Corazon de oro" cultivar. Somatic embryos show growth arrest that is partially overcome by transfer to hormone-free basal medium with activated charcoal. Root induction is possible on basal medium plus 1 mg/L IBA. Plantlets in the greenhouse have variegated leaves true-to-type.

  15. 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.

  16. Cellular and molecular changes associated with somatic embryogenesis induction in Agave tequilana.

    PubMed

    Portillo, L; Olmedilla, A; Santacruz-Ruvalcaba, F

    2012-10-01

    In spite of the importance of somatic embryogenesis for basic research in plant embryology as well as for crop improvement and plant propagation, it is still unclear which mechanisms and cell signals are involved in acquiring embryogenic competence by a somatic cell. The aim of this work was to study cellular and molecular changes involved in the induction stage in calli of Agave tequilana Weber cultivar azul in order to gain more information on the initial stages of somatic embryogenesis in this species. Cytochemical and immunocytochemical techniques were used to identify differences between embryogenic and non-embryogenic cells from several genotypes. Presence of granular structures was detected after somatic embryogenesis induction in embryogenic cells; composition of these structures as well as changes in protein and polysaccharide distribution was studied using Coomassie brilliant blue and Periodic Acid-Schiff stains. Distribution of arabinogalactan proteins (AGPs) and pectins was investigated in embryogenic and non-embryogenic cells by immunolabelling using anti-AGP monoclonal antibodies (JIM4, JIM8 and JIM13) as well as an anti-methyl-esterified pectin-antibody (JIM7), in order to evaluate major modifications in cell wall composition in the initial stages of somatic embryogenesis. Our observations pointed out that induction of somatic embryogenesis produced accumulation of proteins and polysaccharides in embryogenic cells. Presence of JIM8, JIM13 and JIM7 epitopes were detected exclusively in embryogenic cells, which supports the idea that specific changes in cell wall are involved in the acquisition of embryogenic competence of A. tequilana.

  17. Effects of light quality on somatic embryogenesis in Araujia sericifera.

    PubMed

    Torné, Josep M.; Moysset, Luisa; Santos, Mireya; Simón, Esther

    2001-03-01

    The effects of photoperiod, light quality and end-of-day (EOD) phytochrome photoconversion on somatic embryogenesis (SE) of Araujia sericifera petals have been studied. Petals from immature flowers were cultured under 8- and 16-h photoperiods using Gro-lux fluorescent lamps. The photon fluence rate was 90-100 µmol m-2 s-1 and the red (R):far-red (FR) ratio was 98. R, FR, R followed by FR (R-FR) and FR followed by R (FR-R) light treatments were applied for 3 weeks at the end of the photoperiods. In a set of experiments, DL-alpha-difluoromethylarginine (DFMA) or methylglyoxal bis(guanylhydrazone) (MGBG), both inhibitors of polyamine biosynthesis, were added to the culture medium in order to study the involvement of polyamine metabolism. The level of SE was the same in long (LD) and short (SD) days. Thus, the light effect was accomplished after 8 h. All EOD treatments that decreased the Pfr level inhibited SE when applied after SD, but not after LD. The FR-R treatment after LD caused an additional stimulatory effect on SE, even in the presence of polyamine inhibitors. DFMA inhibited SE in both SD and LD, but MGBG did not modify SE in either SD or LD. The R, FR and R-FR treatments did not alter the level of SE when applied after LD in the presence of DFMA or MGBG. However, these treatments decreased SE after SD when the medium contained polyamine inhibitors. Our results suggest that Gro-lux lamps, which produce an extremely high R:FR ratio, promote SE in A. sericifera and a timing response to phytochrome photoconversion during photoperiodic induction. Thus, our data corroborate the involvement of phytochromes and polyamines in SE in A. sericifera, which responded as a light-dominant long-day plant.

  18. 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

  19. 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

  20. Ammonium-related metabolic changes affect somatic embryogenesis in pumpkin (Cucurbita pepo L.).

    PubMed

    Mihaljević, Snježana; Radić, Sandra; Bauer, Nataša; Garić, Rade; Mihaljević, Branka; Horvat, Gordana; Leljak-Levanić, Dunja; Jelaska, Sibila

    2011-11-01

    Somatic embryogenesis in pumpkin can be induced on auxin-containing medium and also on hormone-free medium containing 1mM ammonium (NH(4)(+)) as the sole source of nitrogen. Growth of NH(4)(+)-induced embryogenic tissue was slow and caused considerable acidification of the culture medium. Small spherical cells with dense cytoplasma formed proembryogenic cell clusters that could not develop into late stage embryos. Buffering of NH(4)(+) medium with 25mM 2-(N-morpholino)-ethane-sulfonic acid enhanced tissue proliferation, but no further differentiation was observed. Later stage embryos developed only after re-supply of nitrogen in form of nitrate or l-glutamine. Effects of nitrogen status and pH of culture media on ammonium assimilation were analyzed by following the activity of glutamine synthetase (GS) in relation to phenylalanine ammonia-lyase (PAL). Increased activity of GS and PAL in NH(4)(+) induced tissue coincided with significantly higher activity of stress-related enzymes superoxide dismutase (SOD) and soluble peroxidase (POD), indicating oxidative stress response of embryogenic tissue to NH(4)(+) as the sole source of nitrogen. In addition, considerable increase was observed in callose accumulation and esterase activity, the early markers of somatic embryogenesis. Activity of stress-related enzymes decreased after the re-supply of nitrate (20mM) or Gln (10mM) in combination with NH(4)(+) (1mM), which subsequently triggered globular embryo development. Together, these results suggest that stress responses, as affected by nitrogen supply, contribute to the regulation of embryogenic competence in pumpkin.

  1. 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.

  2. Callose deposition is required for somatic embryogenesis in plasmolyzed Eleutherococcus senticosus zygotic embryos.

    PubMed

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

    2012-10-31

    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.

  3. Callose deposition is required for somatic embryogenesis in plasmolyzed Eleutherococcus senticosus zygotic embryos.

    PubMed

    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

  4. Cellular and molecular changes associated with somatic embryogenesis induction in Agave tequilana.

    PubMed

    Portillo, L; Olmedilla, A; Santacruz-Ruvalcaba, F

    2012-10-01

    In spite of the importance of somatic embryogenesis for basic research in plant embryology as well as for crop improvement and plant propagation, it is still unclear which mechanisms and cell signals are involved in acquiring embryogenic competence by a somatic cell. The aim of this work was to study cellular and molecular changes involved in the induction stage in calli of Agave tequilana Weber cultivar azul in order to gain more information on the initial stages of somatic embryogenesis in this species. Cytochemical and immunocytochemical techniques were used to identify differences between embryogenic and non-embryogenic cells from several genotypes. Presence of granular structures was detected after somatic embryogenesis induction in embryogenic cells; composition of these structures as well as changes in protein and polysaccharide distribution was studied using Coomassie brilliant blue and Periodic Acid-Schiff stains. Distribution of arabinogalactan proteins (AGPs) and pectins was investigated in embryogenic and non-embryogenic cells by immunolabelling using anti-AGP monoclonal antibodies (JIM4, JIM8 and JIM13) as well as an anti-methyl-esterified pectin-antibody (JIM7), in order to evaluate major modifications in cell wall composition in the initial stages of somatic embryogenesis. Our observations pointed out that induction of somatic embryogenesis produced accumulation of proteins and polysaccharides in embryogenic cells. Presence of JIM8, JIM13 and JIM7 epitopes were detected exclusively in embryogenic cells, which supports the idea that specific changes in cell wall are involved in the acquisition of embryogenic competence of A. tequilana. PMID:22270826

  5. 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 μI 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

  6. 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 μI 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.

  7. Comparative proteomic analysis of early somatic and zygotic embryogenesis in Theobroma cacao L.

    PubMed

    Noah, Alexandre Mboene; Niemenak, Nicolas; Sunderhaus, Stephanie; Haase, Christin; Omokolo, Denis Ndoumou; Winkelmann, Traud; Braun, Hans-Peter

    2013-01-14

    Somatic embryogenesis can efficiently foster the propagation of Theobroma cacao, but the poor quality of resulted plantlet hinders the use of this technique in the commercial scale. The current study has been initiated to systematically compare the physiological mechanisms underlying somatic and zygotic embryogenesis in T. cacao on the proteome level. About 1000 protein spots per fraction could be separated by two-dimensional isoelectric focusing/SDS PAGE. More than 50 of the protein spots clearly differed in abundance between zygotic and somatic embryos: 33 proteins spots were at least 3-fold higher in abundance in zygotic embryos and 20 in somatic embryos. Analyses of these protein spots differing in volume by mass spectrometry resulted in the identification of 68 distinct proteins. Many of the identified proteins are involved in genetic information processing (21 proteins), carbohydrate metabolism (11 proteins) and stress response (7 proteins). Somatic embryos especially displayed many stress related proteins, few enzymes involved in storage compound synthesis and an exceptional high abundance of endopeptidase inhibitors. Phosphoenolpyruvate carboxylase, which was accumulated more than 3-fold higher in zygotic embryos, represents a prominent enzyme in the storage compound metabolism in cacao seeds. Implications on the improvement of somatic embryogenesis in cacao are discussed.

  8. ROS Homeostasis Regulates Somatic Embryogenesis via the Regulation of Auxin Signaling in Cotton*

    PubMed Central

    Zhou, Ting; Yang, Xiyan; Guo, Kai; Deng, Jinwu; Xu, Jiao; Gao, Wenhui; Lindsey, Keith; Zhang, Xianlong

    2016-01-01

    Somatic embryogenesis (S.E.) is a versatile model for understanding the mechanisms of plant embryogenesis and a useful tool for plant propagation. To decipher the intricate molecular program and potentially to control the parameters affecting the frequency of S.E., a proteomics approach based on two-dimensional gel electrophoresis (2-DE) combined with MALDI-TOF/TOF was used. A total of 149 unique differentially expressed proteins (DEPs) were identified at different stages of cotton S.E. compared with the initial control (0 h explants). The expression profile and functional annotation of these DEPs revealed that S.E. activated stress-related proteins, including several reactive oxygen species (ROS)-scavenging enzymes. Proteins implicated in metabolic, developmental, and reproductive processes were also identified. Further experiments were performed to confirm the role of ROS-scavenging enzymes, suggesting the involvement of ROS homeostasis during S.E. in cotton. Suppressing the expression of specifically identified GhAPX proteins resulted in the inhibition of dedifferentiation. Accelerated redifferentiation was observed in the suppression lines of GhAPXs or GhGSTL3 in parallel with the alteration of endogenous ascorbate metabolism and accumulation of endogenous H2O2 content. Moreover, disrupting endogenous redox homeostasis through the application of high concentrations of DPI, H2O2, BSO, or GSH inhibited the dedifferentiation of cotton explants. Mild oxidation induced through BSO treatment facilitated the transition from embryogenic calluses (ECs) to somatic embryos. Meanwhile, auxin homeostasis was altered through the perturbation of ROS homeostasis by chemical treatments or suppression of ROS-scavenging proteins, along with the activating/suppressing the transcription of genes related to auxin transportation and signaling. These results show that stress responses are activated during S.E. and may regulate the ROS homeostasis by interacting with auxin signaling

  9. ROS Homeostasis Regulates Somatic Embryogenesis via the Regulation of Auxin Signaling in Cotton.

    PubMed

    Zhou, Ting; Yang, Xiyan; Guo, Kai; Deng, Jinwu; Xu, Jiao; Gao, Wenhui; Lindsey, Keith; Zhang, Xianlong

    2016-06-01

    Somatic embryogenesis (S.E.) is a versatile model for understanding the mechanisms of plant embryogenesis and a useful tool for plant propagation. To decipher the intricate molecular program and potentially to control the parameters affecting the frequency of S.E., a proteomics approach based on two-dimensional gel electrophoresis (2-DE) combined with MALDI-TOF/TOF was used. A total of 149 unique differentially expressed proteins (DEPs) were identified at different stages of cotton S.E. compared with the initial control (0 h explants). The expression profile and functional annotation of these DEPs revealed that S.E. activated stress-related proteins, including several reactive oxygen species (ROS)-scavenging enzymes. Proteins implicated in metabolic, developmental, and reproductive processes were also identified. Further experiments were performed to confirm the role of ROS-scavenging enzymes, suggesting the involvement of ROS homeostasis during S.E. in cotton. Suppressing the expression of specifically identified GhAPX proteins resulted in the inhibition of dedifferentiation. Accelerated redifferentiation was observed in the suppression lines of GhAPXs or GhGSTL3 in parallel with the alteration of endogenous ascorbate metabolism and accumulation of endogenous H2O2 content. Moreover, disrupting endogenous redox homeostasis through the application of high concentrations of DPI, H2O2, BSO, or GSH inhibited the dedifferentiation of cotton explants. Mild oxidation induced through BSO treatment facilitated the transition from embryogenic calluses (ECs) to somatic embryos. Meanwhile, auxin homeostasis was altered through the perturbation of ROS homeostasis by chemical treatments or suppression of ROS-scavenging proteins, along with the activating/suppressing the transcription of genes related to auxin transportation and signaling. These results show that stress responses are activated during S.E. and may regulate the ROS homeostasis by interacting with auxin signaling

  10. Localization and Identification of Phenolic Compounds in Theobroma cacao L. Somatic Embryogenesis

    PubMed Central

    ALEMANNO, L.; RAMOS, T.; GARGADENEC, A.; ANDARY, C.; FERRIERE, N.

    2003-01-01

    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

  11. 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

  12. 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

  13. Loss of CMD2-mediated resistance to cassava mosaic disease in plants regenerated through somatic embryogenesis.

    PubMed

    Beyene, Getu; Chauhan, Raj Deepika; Wagaba, Henry; Moll, Theodore; Alicai, Titus; Miano, Douglas; Carrington, James C; Taylor, Nigel J

    2016-09-01

    Cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) are the two most important viral diseases affecting cassava production in Africa. Three sources of resistance are employed to combat CMD: polygenic recessive resistance, termed CMD1, the dominant monogenic type, named CMD2, and the recently characterized CMD3. The farmer-preferred cultivar TME 204 carries inherent resistance to CMD mediated by CMD2, but is highly susceptible to CBSD. Selected plants of TME 204 produced for RNA interference (RNAi)-mediated resistance to CBSD were regenerated via somatic embryogenesis and tested in confined field trials in East Africa. Although micropropagated, wild-type TME 204 plants exhibited the expected levels of resistance, all plants regenerated via somatic embryogenesis were found to be highly susceptible to CMD. Glasshouse studies using infectious clones of East African cassava mosaic virus conclusively demonstrated that the process of somatic embryogenesis used to regenerate cassava caused the resulting plants to become susceptible to CMD. This phenomenon could be replicated in the two additional CMD2-type varieties TME 3 and TME 7, but the CMD1-type cultivar TMS 30572 and the CMD3-type cultivar TMS 98/0505 maintained resistance to CMD after passage through somatic embryogenesis. Data are presented to define the specific tissue culture step at which the loss of CMD resistance occurs and to show that the loss of CMD2-mediated resistance is maintained across vegetative generations. These findings reveal new aspects of the widely used technique of somatic embryogenesis, and the stability of field-level resistance in CMD2-type cultivars presently grown by farmers in East Africa, where CMD pressure is high.

  14. Regeneration of Solanum nigrum by somatic embryogenesis, involving frog egg-like body, a novel structure.

    PubMed

    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.

  15. 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

  16. Somatic Embryogenesis of Date Palm (Phoenix dactylifera L.) Through Cell Suspension Culture.

    PubMed

    Naik, Poornananda M; Al-Khayri, Jameel M

    2016-01-01

    Date palm (Phoenix dactylifera L.) is the oldest and most economically important plant species distributed in the hot arid regions of the world. Propagation of date palm by seeds produces heterogeneous offspring with inferior field performance and poor fruit quality. Traditionally, date palm is propagated by offshoots, but this method is inefficient for mass propagation because of limited availability of offshoots. Plant regeneration through tissue culture is able to provide technologies for the large-scale propagation of healthy true-to-type plants. The most commonly used technology approach is somatic embryogenesis which presents a great potential for the rapid propagation and genetic resource preservation of this species. Significant progress has been made in the development and optimization of this regeneration pathway through the establishment of embryogenic suspension cultures. This chapter focuses on the methods employed for the induction of callus from shoot tip explants, establishment of cell suspension culture, and subsequent somatic embryogenesis and plant regeneration. PMID:27108330

  17. Somatic embryogenesis of a wild passion fruit species Passiflora cincinnata Masters: histocytological and histochemical evidences.

    PubMed

    Rocha, Diego Ismael; Vieira, Lorena Melo; Tanaka, Francisco André Ossamu; da Silva, Luzimar Campos; Otoni, Wagner Campos

    2012-07-01

    The characterization of cellular changes that occur during somatic embryogenesis is essential for understanding the factors involved in the transition of somatic cells into embryogenically competent cells and determination of cells and/or tissues involved. The present study describes the anatomical and ultrastructural events that lead to the formation of somatic embryos in the model system of the wild passion fruit (Passiflora cincinnata). Mature zygotic embryos were inoculated in Murashige and Skoog induction media supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Zygotic embryo explants at different development stages were collected and processed by conventional methods for studies using light, scanning, and transmission electron microscopy (TEM). Histochemical tests were used to examine the mobilization of reserves. The differentiation of the somatic embryos began in the abaxial side of the cotyledon region. Protuberances were formed from the meristematic proliferation of the epidermal and mesophyll cells. These cells had large nuclei, dense cytoplasm with a predominance of mitochondria, and a few reserve compounds. The protuberances extended throughout the abaxial surface of the cotyledons. The ongoing differentiation of peripheral cells of these structures led to the formation of proembryogenic zones, which, in turn, dedifferentiated into somatic embryos of multicellular origin. In the initial stages of embryogenesis, the epidermal and mesophyll cells showed starch grains and less lipids and protein reserves than the starting explant. These results provide detailed information on anatomical and ultrastructural changes involved in the acquisition of embryogenic competence and embryo differentiation that has been lacking so far in Passiflora.

  18. Endogenous Abscisic Acid and Indole-3-Acetic Acid and Somatic Embryogenesis in Cultured Leaf Explants of Pennisetum purpureum Schum. 1

    PubMed Central

    Rajasekaran, Kanniah; Hein, Mich B.; Vasil, Indra K.

    1987-01-01

    Effects of application in vivo of glyphosate, fluridone, and paclobutrazol to glasshouse-grown donor plants of Pennisetum purpureum Schum. on endogenous levels of abscisic acid (ABA) and indole-3-acetic acid (IAA) in young leaves and on somatic embryogenesis in cultured leaf explants were studied. Treatment of plants with glyphosate (100 milligrams per liter) resulted in elevated levels of endogenous ABA and IAA in young leaves. In contrast, paclobutrazol (50% active ingredient; 200 milligrams per liter) did not alter the endogenous levels of ABA and IAA. Fluridone (100 milligrams per liter) markedly inhibited synthesis of ABA and leaf explants from fluridone-treated plants lost the capacity for somatic embryogenesis. Explants from glyphosate- or paclobutrazol-treated plants did not show any reduction in embryogenic capacity when compared with untreated control plants. Glyphosate and fluridone were also incorporated into the culture media at various concentrations (0 to 20 milligrams per liter) to study their effects in vitro on somatic embryogenesis in leaf explants from untreated, field-grown plants. Glyphosate was inhibitory to somatic embryogenesis but only at concentrations above 5 milligrams per liter. Fluridone inhibited somatic embryogenesis at all concentrations tested. Inhibition of somatic embryogenesis by fluridone, by either in vivo or in vitro application, could be overcome partially by (±)-ABA added to the culture medium. Exogenous application of (±)-ABA enhanced somatic embryogenesis and reduced the formation of nonembryogenic callus. Application of IAA or gibberellic acid (GA3; >5 milligrams per liter) was inhibitory to somatic embryogenesis. These results indicate that endogenous ABA is one of the important factors controlling the embryogenic capacity of leaf explants in Napier grass. PMID:16665403

  19. 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

  20. 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 ...

  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. Genetic transformation via somatic embryogenesis to establish herbicide-resistant opium poppy.

    PubMed

    Facchini, Peter J; Loukanina, Natalia; Blanche, Vincent

    2008-04-01

    A reliable genetic transformation protocol via somatic embryogenesis has been developed for the production of fertile, herbicide-resistant opium poppy plants. Transformation was mediated by Agrobacterium tumefaciens using the pCAMBIA3301 vector, which harbors the phosphinothricin acetyltransferase (pat) gene driven by a tandem repeat of the cauliflower mosaic virus (CaMV) 35S promoter and the beta-glucuronidase (gus) structural gene driven by a single copy of the CaMV 35S promoter between left- and right-border sequences. Co-cultivation of explants and A. tumefaciens was performed in the presence of 50 microM ATP and 50 microM MgCl(2). Root explants pre-cultured on callus induction medium were used for transformation. Herbicide-resistant, proliferating callus was obtained from explants on a medium containing both 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA). Globular embryogenic callus, induced by removal of the BA from the medium, was placed on a hormone-free medium to form somatic embryos, which were converted to plantlets under specific culture conditions. Plantlets with roots were transferred to soil, allowed to mature and set seed. Both pat and gus gene transcripts, and PAT and GUS enzyme activities were detected in the transgenic lines tested. Histochemical localization of GUS activity in T(1) opium poppy plants revealed transgene expression in most tissues of all plant organs. The protocol required 8-12 months to establish transgenic T(1) seed stocks and was developed using a commercial opium poppy cultivar that produces high levels of pharmaceutical alkaloids.

  3. 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.

  4. 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

  5. In vitro propagation of ash (Fraxinus excelsior L.) by somatic embryogenesis.

    PubMed

    Capuana, Maurizio

    2013-01-01

    Induction of somatic embryogenesis is described in common ash (Fraxinus excelsior L.). Embryogenic tissues are obtained from immature zygotic embryos and cultured on a modified Murashige and Skoog (MS) medium containing 8.8 μM 2,4-dichlorophenoxyacetic acid and 4.4 μM benzyl-adenine. Embryogenic tissue is subcultured and multiplied on medium supplemented with reduced concentration of plant growth hormones. Somatic embryos develop and mature by transfer to hormone-free medium and subsequent culture on medium containing low amount of benzyladenine. Somatic embryo germination and conversion are enhanced by cold storage at 4°C and successive transfer onto Woody Plant Medium (WPM). Fully developed plantlets are then transferred to pots and acclimatized in the greenhouse equipped with a mist system. PMID:23179701

  6. 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

  7. Lodgepole pine: the first evidence of seed-based somatic embryogenesis and the expression of embryogenesis marker genes in shoot bud cultures of adult trees.

    PubMed

    Park, So-Young; Klimaszewska, Krystyna; Park, Ji-Young; Mansfield, Shawn D

    2010-11-01

    Of the various alternatives for cloning elite conifers, somatic embryogenesis (SE) appears to be the best option. In recent years, significant areas of lodgepole pine (Pinus contorta) forest have been devastated by the mountain pine beetle (MPB) in Western Canada. In an attempt to establish an SE propagation system for MPB-resistant lodgepole pine, several families displaying varying levels of resistance were selected for experimentation involving shoot bud and immature seed explants. In bud cultures, eight embryogenic lines were induced from 2 of 15 genotypes following various treatments. Genotype had an important influence on embryogenic culture initiation, and this effect was consistent over time. These lines were identified by microscopic observation and genetic markers. Despite the abundance of early somatic embryos, the cultures have yet to develop into mature embryos. In contrast, immature zygotic embryos (ZEs) cultured from megagametophytes initiated SE at an early dominance stage via nodule-type callus in 1 of 10 genotypes. As part of the study, putative embryogenesis-specific genes, WOX2 (WUSCHELL homeobox 2) and HAP3A, were analyzed in cultures of both shoot bud explants and ZEs. On the basis of these analyses, we postulate that PcHAP3A was expressed mainly in callus and may be involved in cell division, whereas WOX2 was expressed mainly in embryonal mass (EM)-like tissues. The findings from this study, based on molecular assessment, suggest that the cell lines derived from bud cultures were truly EM. Moreover, these experimental observations suggest that PcWOX2 could be used as an early genetic marker to discriminate embryogenic cultures from callus. PMID:20935320

  8. Arabinogalactan-proteins stimulate somatic embryogenesis and plant propagation of Pelargonium sidoides.

    PubMed

    Duchow, Stefanie; Dahlke, Renate I; Geske, Thomas; Blaschek, Wolfgang; Classen, Birgit

    2016-11-01

    Root extracts of the medicinal plant Pelargonium sidoides, native to South Africa, are used globally for the treatment of common cold and cough. Due to an increasing economic commercialization of P. sidoides remedies, wild collections of root material should be accompanied by effective methods for plant propagation like somatic embryogenesis. Based on this, the influence of arabinogalactan-proteins (AGPs) on somatic embryogenesis and plant propagation of P. sidoides has been investigated. High-molecular weight AGPs have been isolated from dried roots as well as from cell cultures of P. sidoides with yields between 0.1% and 0.9%, respectively. AGPs are characterized by a 1,3-linked Galp backbone, branched at C6 to 1,6-linked Galp side chains terminated by Araf and to a minor extent by GlcpA, Galp or Rhap. Treatment of explants of P. sidoides with AGPs from roots or suspension culture over 5.5 weeks resulted in effective stimulation of somatic embryo development and plant regeneration. PMID:27516259

  9. 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

  10. Vegetative propagation of Quercus suber L. by somatic embryogenesis. I. Factors affecting the induction in leaves from mature cork oak trees.

    PubMed

    Hernández, I; Celestino, C; Toribio, M

    2003-04-01

    Somatic embryogenesis was induced in expanding leaves from epicormic shoots forced to sprout from segments of branches collected from several hundred-year-old cork oak trees. Following a basic protocol previously defined for leaves taken from seedlings of this species, several factors were studied to improve the response. The induction frequency was significantly higher when the length of exposure to growth regulators was increased from 7 to 30 days. The combined application of NAA and BAP was essential for induction. Although both regulators had a very significant influence, their interaction was not significant, suggesting independent roles. Leaf size had a crucial effect, because beyond a certain threshold, embryogenesis could not be obtained. Embryogenic lines were maintained via repetitive embryogenesis on hormone-free medium for more than 2 years.

  11. 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.

  12. High efficient somatic embryogenesis development from leaf cultures of Citrullus colocynthis (L.) Schrad for generating true type clones.

    PubMed

    Ramakrishna, D; Shasthree, T

    2016-04-01

    We report an efficient somatic embryogenesis and plant regeneration system using leaf cultures of Citrullus colocynthis (L.) and assessed the effect of plant growth regulators on the regeneration process. Initially leaf explants were cultured on Murashige and Skoog medium supplemented with different concentrations of auxins viz., 2,4-dichlorophenoxyacetic acid, 1-naphthaleneacetic acid, gibberellic acid alone and along with combination of 6-benzylaminopurine. The different forms of calli such as compact, white friable, creamy friable, brownish nodular, green globular and green calli were induced from the leaf explants on MS medium containing different concentrations of auxins and gibberellins. Subsequently initial callus was subcultured at 1.5 mg L(-1) BAP + 1.0 mg L(-1) 2,4-D which resulted in 25 % somatic embryos from 85 % nodular embryogenic nodular callus that is highest percentage. Similarly the lowest percentage of somatic embryos was recorded at 2.5 mg L(-1) BAP + 0.5 mg L(-1) NAA from 55 % embryogenic globular callus i.e., 16 %. High frequency of embryo development takes place at intermittent light when compared with continuous light in the individual subcultures. The cotyledonary embryos were developed into complete platelets on MS medium. In vitro regenerated plantlets were washed to remove the traces of agar and then transferred to sterile vermiculite and sand (2:1) containing pot.

  13. High efficient somatic embryogenesis development from leaf cultures of Citrullus colocynthis (L.) Schrad for generating true type clones.

    PubMed

    Ramakrishna, D; Shasthree, T

    2016-04-01

    We report an efficient somatic embryogenesis and plant regeneration system using leaf cultures of Citrullus colocynthis (L.) and assessed the effect of plant growth regulators on the regeneration process. Initially leaf explants were cultured on Murashige and Skoog medium supplemented with different concentrations of auxins viz., 2,4-dichlorophenoxyacetic acid, 1-naphthaleneacetic acid, gibberellic acid alone and along with combination of 6-benzylaminopurine. The different forms of calli such as compact, white friable, creamy friable, brownish nodular, green globular and green calli were induced from the leaf explants on MS medium containing different concentrations of auxins and gibberellins. Subsequently initial callus was subcultured at 1.5 mg L(-1) BAP + 1.0 mg L(-1) 2,4-D which resulted in 25 % somatic embryos from 85 % nodular embryogenic nodular callus that is highest percentage. Similarly the lowest percentage of somatic embryos was recorded at 2.5 mg L(-1) BAP + 0.5 mg L(-1) NAA from 55 % embryogenic globular callus i.e., 16 %. High frequency of embryo development takes place at intermittent light when compared with continuous light in the individual subcultures. The cotyledonary embryos were developed into complete platelets on MS medium. In vitro regenerated plantlets were washed to remove the traces of agar and then transferred to sterile vermiculite and sand (2:1) containing pot. PMID:27436919

  14. 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

  15. AGAMOUS-Like15 Promotes Somatic Embryogenesis in Arabidopsis and Soybean in Part by the Control of Ethylene Biosynthesis and Response1[C][W][OA

    PubMed Central

    Zheng, Qiaolin; Zheng, Yumei; Perry, Sharyn E.

    2013-01-01

    Many of the regulatory processes occurring during plant embryogenesis are still unknown. Relatively few cells are involved, and they are embedded within maternal tissues, making this developmental phase difficult to study. Somatic embryogenesis is a more accessible system, and many important regulatory genes appear to function similar to zygotic development, making somatic embryogenesis a valuable model for the study of zygotic processes. To better understand the role of the Arabidopsis (Arabidopsis thaliana) MADS factor AGAMOUS-Like15 (AGL15) in the promotion of somatic embryogenesis, direct target genes were identified by chromatin immunoprecipitation-tiling arrays and expression arrays. One potential directly up-regulated target was At5g61590, which encodes a member of the ethylene response factor subfamily B-3 of APETALA2/ETHYLENE RESPONSE FACTOR transcription factors and is related to Medicago truncatula SOMATIC EMBRYO-RELATED FACTOR1 (MtSERF1), which has been shown to be required for somatic embryogenesis in M. truncatula. Here, we report confirmation that At5g61590 is a directly expressed target of AGL15 and that At5g61590 is essential for AGL15’s promotion of somatic embryogenesis. Because At5g61590 is a member of the ETHYLENE RESPONSE FACTOR family, effects of ethylene on somatic embryogenesis were investigated. Precursors to ethylene stimulate somatic embryogenesis, whereas inhibitors of ethylene synthesis or perception reduce somatic embryogenesis. To extend findings to a crop plant, we investigated the effects of ethylene on somatic embryogenesis in soybean (Glycine max). Furthermore, we found that a potential ortholog of AGL15 in soybean (GmAGL15) up-regulates ethylene biosynthesis and response, including direct regulation of soybean orthologs of At5g61590/MtSERF1 named here GmSERF1 and GmSERF2, in concordance with the M. truncatula nomenclature. PMID:23457229

  16. GhAGL15s, preferentially expressed during somatic embryogenesis, promote embryogenic callus formation in cotton (Gossypium hirsutum L.).

    PubMed

    Yang, Zuoren; Li, Changfeng; Wang, Ye; Zhang, Chaojun; Wu, Zhixia; Zhang, Xueyan; Liu, Chuanliang; Li, Fuguang

    2014-10-01

    Somatic embryogenesis is a useful tool for gene transfer and propagation of plants. AGAMOUS-LIKE15 (AGL15) promotes somatic embryogenesis in many plant species. In this study, three homologous AGL15 genes were isolated from Gossypium hirsutum L., namely GhAGL15-1, GhAGL15-3, and GhAGL15-4. Their putative proteins contained a highly conserved MADS-box DNA-binding domain and a less conserved K domain. Phylogenetic analysis suggested that the three GhAGL15s clustered most closely with AGL15 proteins in other plants. Subcellular location analyses revealed that three GhAGL15s were localized in the nucleus. Furthermore, their expression levels increased following embryogenic callus induction, but sharply decreased during the embryoid stage. GhAGL15-1 and GhAGL15-3 were significantly induced by 2,4-D and kinetin, whereas GhAGL15-4 was only responsive to 2,4-D treatment. Over-expression of the three GhAGL15s in cotton callus improved callus quality and significantly increased the embryogenic callus formation rate, while GhAGL15-4 had the highest positive effect on the embryogenic callus formation rate (an increase from 38.1 to 65.2%). These results suggest that over-expression of GhAGL15s enhances embryogenic potential of transgenic calli. Therefore, spatiotemporal manipulation of GhAGL15s expression may prove valuable in improving cotton transformation efficiency.

  17. Vegetative propagation of Quercus suber L. by somatic embryogenesis. II. Plant regeneration from selected cork oak trees.

    PubMed

    Hernández, I; Celestino, C; Alegre, J; Toribio, M

    2003-04-01

    The regeneration of somatic seedlings from selected 100-year-old cork oak trees is reported. The induction of somatic embryogenesis from leaves of epicormic shoots was significantly affected by genotype, harvesting time and their interaction. Leaves from all five selected trees produced somatic embryos when the segments of branches used as sources of epicormic shoots were collected in May. Genotype, but not the level of photosynthetically active radiation, affected the proliferation of the embryogenic lines and the number of detachable embryos that could be obtained from them. Genotype also affected several steps leading to conversion of somatic embryos, from germination to complete acclimatisation of somatic seedlings. Almost 40% of the somatic embryos from all lines germinated, showing coordinated root and shoot growth. Although the mean percentage of recovery for the whole process was low, plants could be regenerated from four of the five trees tested.

  18. Somatic Embryogenesis in Olive (Olea europaea L. subsp. europaea var. sativa and var. sylvestris).

    PubMed

    Rugini, Eddo; Silvestri, Cristian

    2016-01-01

    Protocols for olive somatic embryogenesis from zygotic embryos and mature tissues have been described for both Olea europaea sub. europaea var. sativa and var. sylvestris. Immature zygotic embryos (no more than 75 days old), used after fruit collection or stored at 12-14 °C for 2-3 months, are the best responsive explants and very slightly genotype dependent, and one single protocol can be effective for a wide range of genotypes. On the contrary, protocols for mature zygotic embryos and for mature tissue of cultivars are often genotype specific, so that they may require many adjustments according to genotypes. The use of thidiazuron and cefotaxime seems to be an important trigger for induction phase particularly for tissues derived from cultivars. Up to now, however, the application of this technique for large-scale propagation is hampered also by the low rate of embryo germination; it proves nonetheless very useful for genetic improvement.

  19. Characterization and expression analysis of somatic embryogenesis receptor-like kinase genes from Phalaenopsis.

    PubMed

    Huang, Y W; Tsai, Y J; Chen, F C

    2014-12-18

    Somatic embryogenesis receptor-like kinase (SERK) genes have been found to be involved in the somatic embryogenesis of several plant species. We identified and characterized 5 PhSERK genes in the Phalaenopsis orchid. The amino acid sequences of PhSERKs and other SERK proteins are highly conserved, with the highest homology observed in the leucine-rich repeat-receptor-like kinase domain. All 5 PhSERKs were expressed in all Phalaenopsis organs examined (root, leaf, shoot apical meristem, and flower), with the strongest expression, particularly for PhSERK1 and 3, in the shoot apical meristem of mature plants. Expression of all PhSERKs was downregulated during early floral bud development and was upregulated gradually until the semi-open flower stage was reached. All 5 PhSERKs were expressed during both seed germination and protocorm-like-body (PLB) development. In germinated seeds, quantitative real-time PCR revealed upregulation of all PhSERKs except PhSERK4 at 1 week and downregulation after 4 weeks. The 5 PhSERKs were differentially expressed in the early stage of PLB development and maintained substantial levels during PLB formation, with PhSERK1 and 5 upregulated 1 week after culture and PhSERK2, 3, and 4 downregulated over this period. Because physical wounding of PLB stimulates secondary PLB formation, the PhSERK5 expression peak at week 3 coincided with visible and fully developed secondary PLBs. PhSERK5 may be important in PLB induction and subsequent development. Our PhSERK expression analysis revealed that these genes have a broad role during orchid plant development.

  20. Loss of CMD2‐mediated resistance to cassava mosaic disease in plants regenerated through somatic embryogenesis

    PubMed Central

    Chauhan, Raj Deepika; Wagaba, Henry; Moll, Theodore; Alicai, Titus; Miano, Douglas; Carrington, James C.; Taylor, Nigel J.

    2016-01-01

    Summary Cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) are the two most important viral diseases affecting cassava production in Africa. Three sources of resistance are employed to combat CMD: polygenic recessive resistance, termed CMD1, the dominant monogenic type, named CMD2, and the recently characterized CMD3. The farmer‐preferred cultivar TME 204 carries inherent resistance to CMD mediated by CMD2, but is highly susceptible to CBSD. Selected plants of TME 204 produced for RNA interference (RNAi)‐mediated resistance to CBSD were regenerated via somatic embryogenesis and tested in confined field trials in East Africa. Although micropropagated, wild‐type TME 204 plants exhibited the expected levels of resistance, all plants regenerated via somatic embryogenesis were found to be highly susceptible to CMD. Glasshouse studies using infectious clones of East African cassava mosaic virus conclusively demonstrated that the process of somatic embryogenesis used to regenerate cassava caused the resulting plants to become susceptible to CMD. This phenomenon could be replicated in the two additional CMD2‐type varieties TME 3 and TME 7, but the CMD1‐type cultivar TMS 30572 and the CMD3‐type cultivar TMS 98/0505 maintained resistance to CMD after passage through somatic embryogenesis. Data are presented to define the specific tissue culture step at which the loss of CMD resistance occurs and to show that the loss of CMD2‐mediated resistance is maintained across vegetative generations. These findings reveal new aspects of the widely used technique of somatic embryogenesis, and the stability of field‐level resistance in CMD2‐type cultivars presently grown by farmers in East Africa, where CMD pressure is high. PMID:26662210

  1. Histological analysis of indirect somatic embryogenesis in the Marsh clubmoss Lycopodiella inundata (L.) Holub (Pteridophytes).

    PubMed

    Atmane; Blervacq; Michaux-Ferriere; Vasseur

    2000-07-28

    An efficient in vitro plant regeneration method was developed for Lycopodiella inundata (L.) Holub, an endangered medicinal Lycopod (Pteridophytes). Vegetative apices were used as explant material. Nodular calluses were established after three cycles (13 weeks each) on a medium containing a few minerals and organic compounds and supplemented with 0.05 µM 3-indolebutyric acid (IBA) and 1.4 µM kinetin (Kin). Propagation was achieved every 13 weeks on this callus medium (CM). When nodular calluses were transferred on a medium supplemented with 2.5 µM IBA and 0.33 µM gibberellic acid (GA(3)) designated as embryogenic medium (EM), organized structures appeared and developed into plantlets. Development phases were characterized by histological studies. Some phases of zygotic embryogenesis previously described for Lycopods were observed in L. inundata. Histological analyses established that an indirect somatic embryo was derived from a single embryogenic cell by following the zygotic developmental pathway. As this phenomenon has not previously been reported in Lycopods, a comparison between somatic and zygotic embryos is discussed based upon morphology and histology. PMID:10936522

  2. 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.

  3. Identification of a novel factor, vanillyl benzyl ether, which inhibits somatic embryogenesis of Japanese larch (Larix leptolepis Gordon).

    PubMed

    Umehara, Mikihisa; Ogita, Shinjiro; Sasamoto, Hamako; Koshino, Hiroyuki; Asami, Tadao; Fujioka, Shozo; Yoshida, Shigeo; Kamada, Hiroshi

    2005-03-01

    In contrast to angiosperms, some gymnosperms form well-developed suspensors in somatic embryogenesis. This characteristic makes it easy to study suspensor biology. In cultures with high cell densities, somatic embryogenesis of Japanese larch, especially the suspensor development, is strongly inhibited due to factor(s) that are released by the cells into the culture medium. In this study, we purified and identified one of the inhibitory factors present in high-cell-density conditioned medium (HCM) of larch cells. The factor with the strongest inhibitory activity was purified by dialysis, extraction by ethyl acetate, octadecylsilyl (ODS) column chromatography and high-performance liquid chromatography (HPLC). The inhibitory factor was identified as vanillyl benzyl ether (VBE) by physicochemical analysis. This compound was first isolated from natural resources. Authentic VBE inhibited somatic embryo formation in Japanese larch, and the inhibitory effect in the suspensor was stronger than in the embryo proper. Furthermore, quantification of VBE by HPLC demonstrated that VBE accumulates at high concentrations in HCM. These results suggest that VBE is a novel negative regulator of somatic embryogenesis.

  4. Somatic embryogenesis in pumpkin (Cucurbita pepo L.): control of somatic embryo development by nitrogen compounds.

    PubMed

    Leljak-Levanić, Dunja; Bauer, Natasa; Mihaljević, Snjezana; Jelaska, Sibila

    2004-02-01

    Embryogenic cultures of pumpkin (Cucurbita pepo L.) were initiated from mechanically wounded mature zygotic embryos on 2,4-D-containing MS medium, and on hormone-free, semisolid modified MS medium containing NH4Cl as the sole source of nitrogen. The habituated line was derived from the embryogenic tissue induced with 2,4-D and maintained on medium without growth regulators. Sustained subculturing of the three embryogenic lines on a medium with NH4Cl as the sole source of nitrogen enabled the establishment of highly uniform cultures in which no further development into mature embryo stages occurred. The tissue consisting of proembryogenic globules or globular stage embryos was maintained, without decline, for over six years. Globular embryos proceeded to maturity when a combination of reduced (NH4) and unreduced (NO3) forms of nitrogen was provided in the medium. Different nitrogen sources in the medium caused changes of medium pH during subculture in the pH range of 4.0-6.5. The tissue growth and embryo development were blocked on medium with pH adjusted and stabilized at 4.0 or at 3.2.

  5. Proteomic analysis of somatic embryogenesis in Valencia sweet orange (Citrus sinensis Osbeck).

    PubMed

    Pan, Zhiyong; Guan, Rui; Zhu, Shiping; Deng, Xiuxin

    2009-02-01

    Two dimensional gel electrophoresis combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed to study the somatic embryogenesis (SE) in Valencia sweet orange (Citrus sinensis Osbeck). Twenty-four differentially expressed proteins were identified at five time points of citrus SE (0, 1, 2, 3, 4 weeks after embryo initiation) covering globular, heart/torpedo and cotyledon-shaped embryo stages. The general expression patterns for these proteins were consistent with those appeared at 4 weeks of citrus SE. The most striking feature of our study was that five proteins were predicted to be involved in glutathione (GSH) metabolism and anti-oxidative stress, and they exhibited different expression patterns during SE. Based on that oxidative stress has been validated to enhance SE, the preferential representation for anti-oxidative proteins suggests that they could have a developmental role in citrus SE. Some proteins involved in cell division, photosynthesis and detoxification were also identified, and their possible roles in citrus SE were discussed.

  6. 2,4,5-Trichlorophenoxyacetic acid promotes somatic embryogenesis in the rose cultivar "Livin' Easy" (Rosa sp.).

    PubMed

    Estabrooks, Tammy; Browne, Robin; Dong, Zhongmin

    2007-02-01

    Somatic embryogenesis (SE) offers vast potential for the clonal propagation of high-value roses. However, some recalcitrant cultivars unresponsive to commonly employed SE-inducing agents and low induction rates currently hinder the commercialization of SE technology in rose. Rose SE technology requires improvement before it can be implemented as a production system on a commercial scale. In the present work, we assessed 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), a synthetic auxin not previously tested in rose, for its effectiveness to induce SE in the rose cultivar "Livin' Easy" (Rosa sp.). We ran a parallel comparison to the commonly used 2,4-dichlorophenoxyacetic acid (2,4-D). We tested each auxin with two different basal media: Murashige and Skoog (MS) basal medium and woody plant medium (WPM). MS medium resulted in somatic embryo production, whereas WPM did not. 2,4,5-T induced SE over a greater concentration range than 2,4-D's and resulted in significantly greater embryo yields. 2,4,5-T at a concentration of 10 or 25 microM was better for embrygenic tissue initiation than 2,4,5-T at 5 microM. Further embryo development occurred when the tissue was transferred to plant growth regulator (PGR) free medium or media with 40% the original auxin concentration. However, the PGR-free medium resulted in a high percentage of abnormal embryos (32.31%) compared to the media containing auxins. Upon transfer to germination medium, somatic embryos successfully converted into plantlets at rates ranging from 33.3 to 95.2%, depending on treatment. Survival rates 3 months ex vitro averaged 14.0 and 55.6% for 2,4-D- and 2,4,5-T-derived plantlets, respectively. Recurrent SE was observed in 60.2% of the plantlets growing on germination medium. This study is the first report of SE in the commercially valuable rose cultivar 'Livin' Easy' (Rosa sp.) and a suitable methodology was developed for SE of this rose cultivar.

  7. Pretreatments, conditioned medium and co-culture increase the incidence of somatic embryogenesis of different Cichorium species

    PubMed Central

    Couillerot, Jean-Paul; Windels, David; Vazquez, Franck; Michalski, Jean-Claude; Hilbert, Jean-Louis; Blervacq, Anne-Sophie

    2012-01-01

    Somatic embryogenesis (SE) in Cichorium involves dedifferentiation and redifferentiation of single cells and can be induced by specific in vitro culture conditions. We have tested the effect of various treatments on the incidence of SE (ISE) of an interspecific embryogenic hybrid (C. endivia x C. intybus) and of different commercial chicories (C. endivia and C. intybus) that are typically recalcitrant to SE in standard culture conditions. We found that the ISE of the hybrid is significantly increased by pretreatment of tissues by submersion in solutions of glycerol, abscisic acid, spermine, putrescine or of combinations of these compounds. Interestingly, the most efficient of these pretreatments also had an unexpectedly high effect on the ISE of the C. intybus cultivars. The ISE of the hybrid and of the commercial chicories were increased when explants were co-cultured with highly embryogenic chicory explants or when they were cultured in conditioned medium. These observations established that unidentified SE-promoting factors are released in the culture medium. HPLC analyses of secreted Arabino-Galactan Proteins (AGPs), which are known to stimulate SE, did not allow identifying a fraction containing differentially abundant AGP candidates. However, pointing to their role in promoting SE, we found that the hybrid had a drastically higher ISE when amino sugars and L-Proline, the putative precursors of secreted AGPs, were both added to the medium. PMID:22301978

  8. 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

  9. Annotation of differentially expressed genes in the somatic embryogenesis of musa and their location in the banana genome.

    PubMed

    Maldonado-Borges, Josefina Ines; Ku-Cauich, José Roberto; Escobedo-Graciamedrano, Rosa Maria

    2013-01-01

    Analysis of cDNA-AFLP was used to study the genes expressed in zygotic and somatic embryogenesis of Musa acuminata Colla ssp. malaccensis, and a comparison was made between their differential transcribed fragments (TDFs) and the sequenced genome of the double haploid- (DH-) Pahang of the malaccensis subspecies that is available in the network. A total of 253 transcript-derived fragments (TDFs) were detected with apparent size of 100-4000 bp using 5 pairs of AFLP primers, of which 21 were differentially expressed during the different stages of banana embryogenesis; 15 of the sequences have matched DH-Pahang chromosomes, with 7 of them being homologous to gene sequences encoding either known or putative protein domains of higher plants. Four TDF sequences were located in all Musa chromosomes, while the rest were located in one or two chromosomes. Their putative individual function is briefly reviewed based on published information, and the potential roles of these genes in embryo development are discussed. Thus the availability of the genome of Musa and the information of TDFs sequences presented here opens new possibilities for an in-depth study of the molecular and biochemical research of zygotic and somatic embryogenesis of Musa.

  10. Involvement of peroxidase activity in developing somatic embryos of Medicago arborea L. Identification of an isozyme peroxidase as biochemical marker of somatic embryogenesis.

    PubMed

    Gallego, Piedad; Martin, Luisa; Blazquez, Antonio; Guerra, Hilario; Villalobos, Nieves

    2014-01-15

    The legume Medicago arborea L. is very interesting as regards the regeneration of marginal arid soils. The problem is that it does not have a good germinative yield. It was therefore decided to regenerate via somatic embryogenesis and find a marker of embryogenic potential. In this study, peroxidase activity was evaluated in non-embryogenic and embryogenic calli from M. arborea L. A decrease in soluble peroxidase activity is observed in its embryonic calli at the time at which the somatic embryos begin to appear. This activity is always lower in embryonic calli than in non-embryonic ones (unlike what happens in the case of wall-bound peroxidases). These results suggest that peroxidases can be considered to be enzymes involved in somatic embryogenesis in M. arborea. In addition, isozyme analyses were carried out on protein extracts using polyacrylamide gel electrophoresis. The band called P5 was detected only in embryogenic cultures at very early stages of development. This band was digested with trypsin and analyzed using linear ion trap (LTQ) mass spectrometer. In P5 isoform a peroxidase-L-ascorbate peroxidase was identified. It can be used as a marker that allows the identification of embryological potential.

  11. 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.

  12. Developmental and hormonal regulation of direct shoot organogenesis and somatic embryogenesis in sugarcane (Saccharum spp. interspecific hybrids) leaf culture.

    PubMed

    Lakshmanan, Prakash; Geijskes, R Jason; Wang, Lifang; Elliott, Adrian; Grof, Christopher P L; Berding, Nils; Smith, Grant R

    2006-10-01

    Rapid and efficient in vitro regeneration methods that minimise somaclonal variation are critical for the genetic transformation and mass propagation of commercial varieties. Using a transverse thin cell layer culture system, we have identified some of the developmental and physiological constraints that limit high-frequency regeneration in sugarcane leaf tissue. Tissue polarity and consequently the orientation of the explant in culture, size and developmental phase of explant, and auxin concentration play a significant role in determining the organogenic potential of leaf tissue in culture. Both adventitious shoot production and somatic embryogenesis occurred on the proximal cut surface of the explant, and a regeneration gradient, decreasing gradually from the basal to the distal end, exists in the leaf roll. Importantly, auxin, when added to the culture medium, reduced this spatial developmental constraint, as well as the effect of genotype on plant regeneration. Transverse sections (1-2 mm thick) obtained from young leaf spindle rolls and orienting explants with its distal end facing the medium (directly in contact with medium) are critical for maximum regeneration. Shoot regeneration was observed as early as 3 weeks on MS medium supplemented with alpha-naphthalenencetic acid (NAA) and 6-benzyladenine, while somatic embryogenesis or both adventitious shoot organogenesis and somatic embryogenesis occurred on medium with NAA and chlorophenoxyacetic acid. Twenty shoots or more could be generated from a single transverse section explant. These shoots regenerated roots and successfully established after transplanted to pots. Large numbers of plantlets can be regenerated directly and rapidly using this system. SmartSett, the registered name for this process and the plants produced, will have significant practical applications for the mass propagation of new cultivars and in genetic modification programs. The SmartSett system has already been used commercially to

  13. Influences of aging and cloning methods on the capacity for somatic embryogenesis of a mature Hevea brasiliensis genotype.

    PubMed

    Lardet, Ludovic; Dessailly, Florence; Carron, Marc-Philippe; Montoro, Pascal; Monteuuis, Olivier

    2009-02-01

    We compared embryogenic capacities of integument explants excised from three sources of the Hevea brasiliensis (Müll. Arg.) mature genotype PB 260. The three sources were 17-year-old (BT 86) and 7-year-old (BT 96) budded trees and 7-year-old emblings (EM 96). The highest proportions of embryogenic calluses obtained from the total number of integument explants initially used were from trees of EM 96 origin, followed by BT 96 trees, with explants from BT 86 trees producing the lowest number of embryogenic calluses. Further initiation of embryogenic callus lines from the primary somatic embryos derived from the three sources was successful only for EM 96. Somatic embryo cultures from BT 86 and BT 96 sources produced only friable calluses that could not be further amplified. Overall, somatic embryo explants derived from EM 96 responded over a wider range of 3,4-dichlorophenoxyacetic acid and kinetin concentrations than the somatic embryo explants from BT 86 and BT 96 origins. The effects of chronologic, ontogenetic and physiologic aging on explant capacity for somatic embryogenesis and on the overall efficiency of the process in H. brasiliensis are discussed. PMID:19203954

  14. Somatic embryogenesis and synthetic seed production--a biotechnological approach for true-to-type propagation and in vitro conservation of an ornamental bulbaceous plant Drimiopsis kirkii Baker.

    PubMed

    Haque, Sk Moquammel; Ghosh, Biswajit

    2014-04-01

    An efficient plant regeneration protocol through indirect somatic embryogenesis pathway via callus had been developed from the leaf explant of an ornamental bulbaceous plant Drimiopsis kirkii. Optimum friable calli were induced on Murashige and Skoog (MS) basal medium supplemented with 3.0 mg/l of 2,4-dichlorophenoxyacetic acid and 1.0 mg/l of α-naphthalene acetic acid (NAA). On subculturing the callus on MS medium supplemented with 2.5 mg/l of thidiazuron (TDZ), 73.3 % of the cultures responded with 20.4 ± 0.3 somatic embryos (SEs) per 500 mg callus at different stages of development after 6 weeks of culture. The highest response of 86.7 % with 28.3 ± 0.5 embryos per 500 mg callus was observed on MS medium supplemented with 2.5 mg/l TDZ and 1.0 mg/l NAA. SEs were encapsulated in calcium alginate beads for the production of synthetic seeds (SSs) and their storability was investigated. The highest SS germination (93.3 %) was observed in 1.0 % sodium alginate followed by 86.7 % germination with 2.5 % sodium alginate. The SSs were stored at three different temperatures (4, 15, and 24 ºC) up to 6 months. The SSs kept at 15 °C showed 64.4 % germinability even after 4 months of storage. Both nonencapsulated and encapsulated SE-derived plants were successfully transferred to soil with 93.3 and 88.3 % survival rate accordingly. Randomly amplified polymorphic DNA (RAPD) analysis revealed that there were no somaclonal variations among the plants produced via somatic embryogenesis and they are true-to-type to their parental plant. These results confirmed the most reliable methods, which can be further used for genetic transformation studies as well as for mass propagation of ornamental D. kirkii at a commercial level. PMID:24604129

  15. 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

  16. Plant regeneration from protoplasts ofVicia narbonensis via somatic embryogenesis and shoot organogenesis.

    PubMed

    Tegeder, M; Kohn, H; Nibbe, M; Schieder, O; Pickardt, T

    1996-11-01

    Protoplasts ofVicia narbonensis isolated from epicotyls and shoot tips of etiolated seedlings were embedded in 1.4% sodium-alginate at a final density of 2.5×10(5) protoplasts/ml and cultivated in Kao and Michayluk-medium containing 0.5 mg/I of each of 2,4- dichlorophenoxyacetic acid, naphthylacetic acid and 6 -benzylaminopurine. A division frequency of 36% and a plating efficiency of 0.40-0.5% were obtained. Six weeks after embedding, protoplast-derived calluses were transferred onto gelrite-solidified Murashige and Skoog-media containing various growth regulators. Regeneration of plants was achieved via two morphologically distinguishable pathways. A two step protocol (initially on medium with a high auxin concentration followed by a culture phase with lowered auxin amount) was used to regenerate somatic embryos, whereas cultivation on medium containing thidiazuron and naphthylacetic acid resulted in shoot morphogenesis. Mature plants were recovered from both somatic embryos as well as from thidiazuron-induced shoots.

  17. In vitro regeneration through organogenesis and somatic embryogenesis in pigeon pea [ Cajanus cajan (L.) Millsp.] cv. JKR105.

    PubMed

    Krishna, Gaurav; Reddy, P Sairam; Ramteke, Pramod W; Rambabu, Pogiri; Sohrab, Sayed S; Rana, Debashis; Bhattacharya, Parthasarathi

    2011-10-01

    In vitro regeneration of pigeon pea through organogenesis and somatic embryogenesis was demonstrated with pigeon pea cv. JKR105. Embryonic axes explants of pigeon pea showed greater regeneration of shoot buds on 2.5 mg L(-1) 6-benzylaminopurine (BAP) in the medium, followed by further elongation at lower concentrations. Rooting of shoots was observed on half-strength Murashige and Skoog (MS) medium with 2 % sucrose and 0.5 mg L(-1) 3-indolebutyric acid (IBA). On the other hand, the regeneration of globular embryos from cotyledon explant was faster and greater with thidiazuron (TDZ) than BAP with sucrose as carbohydrate source. These globular embryos were maturated on MS medium with abscisic acid (ABA) and finally germinated on half-strength MS medium at lower concentrations of BAP. Comparison of regeneration pathways in pigeon pea cv. JKR105 showed that the turnover of successful establishment of plants achieved through organogenesis was more compared to somatic embryogenesis, despite the production of more embryos than shoot buds. PMID:23573031

  18. In vitro regeneration through organogenesis and somatic embryogenesis in pigeon pea [ Cajanus cajan (L.) Millsp.] cv. JKR105.

    PubMed

    Krishna, Gaurav; Reddy, P Sairam; Ramteke, Pramod W; Rambabu, Pogiri; Sohrab, Sayed S; Rana, Debashis; Bhattacharya, Parthasarathi

    2011-10-01

    In vitro regeneration of pigeon pea through organogenesis and somatic embryogenesis was demonstrated with pigeon pea cv. JKR105. Embryonic axes explants of pigeon pea showed greater regeneration of shoot buds on 2.5 mg L(-1) 6-benzylaminopurine (BAP) in the medium, followed by further elongation at lower concentrations. Rooting of shoots was observed on half-strength Murashige and Skoog (MS) medium with 2 % sucrose and 0.5 mg L(-1) 3-indolebutyric acid (IBA). On the other hand, the regeneration of globular embryos from cotyledon explant was faster and greater with thidiazuron (TDZ) than BAP with sucrose as carbohydrate source. These globular embryos were maturated on MS medium with abscisic acid (ABA) and finally germinated on half-strength MS medium at lower concentrations of BAP. Comparison of regeneration pathways in pigeon pea cv. JKR105 showed that the turnover of successful establishment of plants achieved through organogenesis was more compared to somatic embryogenesis, despite the production of more embryos than shoot buds.

  19. 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

  20. [Regulation of somatic embryogenesis in Panax ginseng C. A. Meyer cell cultures by PgCDPK2DS1].

    PubMed

    Shumakova, O A; Kiselev, K V

    2014-06-01

    We isolated the full-length cDNA of PgCDPK2DS1 gene, the expression of which was significantly increased at early stages of embryo development in cell cultures of ginseng P. ginseng 2c3. Interest in this gene also was supported by its nonstandard structure: the amino acid sequence of the PgCDPK2DS1 gene contained only the N-terminal domain and 80% of the kinase domain. Overexpression of the PgCDPK2DS1 gene in nonembryonic calli 1c resulted in the appearance of embryonic structures in the PgCDPK2DS1-transgenic ginseng cell culture 1c-2d. Also, expression of the plant embryogenesis marker genes WUS and SERK significantly increased in cell culture 1c-2d. The observed embryo-like structures were at early stages of embryo development; attempts to obtain an adult plant from these embryo-like structures were unsuccessful. Overexpression of PgCDPK2DS1 gene in the embryonic cell culture PG resulted in a decrease of embryonic structures in the PgCDPK2DS1-transgenic ginseng cell culture PG-2d. Moreover, expression of the plant embryogenesis marker genes WUS and SERK and expression of the endogenous PgCDPK2DS1 significantly decreased in the cell culture PG-2d. Thus, for the first time it was shown that the PgCDPK2DS1 gene is involved in the regulation of somatic embryogenesis in P. ginseng cell cultures.

  1. Effect of Cryopreservation and Post-Cryopreservation Somatic Embryogenesis on the Epigenetic Fidelity of Cocoa (Theobroma cacao L.)

    PubMed Central

    Adu-Gyamfi, Raphael; Wetten, Andy; Marcelino Rodríguez López, Carlos

    2016-01-01

    While cocoa plants regenerated from cryopreserved somatic embryos can demonstrate high levels of phenotypic variability, little is known about the sources of the observed variability. Previous studies have shown that the encapsulation-dehydration cryopreservation methodology imposes no significant extra mutational load since embryos carrying high levels of genetic variability are selected against during protracted culture. Also, the use of secondary rather than primary somatic embryos has been shown to further reduce the incidence of genetic somaclonal variation. Here, the effect of in vitro conservation, cryopreservation and post-cryopreservation generation of somatic embryos on the appearance of epigenetic somaclonal variation were comparatively assessed. To achieve this we compared the epigenetic profiles, generated using Methylation Sensitive Amplified Polymorphisms, of leaves collected from the ortet tree and from cocoa somatic embryos derived from three in vitro conditions: somatic embryos, somatic embryos cryopreserved in liquid nitrogen and somatic embryos generated from cryoproserved somatic embryos. Somatic embryos accumulated epigenetic changes but these were less extensive than in those regenerated after storage in LN. Furthermore, the passage of cryopreserved embryos through another embryogenic stage led to further increase in variation. Interestingly, this detected variability appears to be in some measure reversible. The outcome of this study indicates that the cryopreservation induced phenotypic variability could be, at least partially, due to DNA methylation changes. Key message: Phenotypic variability observed in cryostored cocoa somatic-embryos is epigenetic in nature. This variability is partially reversible, not stochastic in nature but a directed response to the in-vitro culture and cryopreservation. PMID:27403857

  2. 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

  3. 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.

  4. 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

  5. De novo transcriptome analysis reveals insights into dynamic homeostasis regulation of somatic embryogenesis in upland cotton (G. hirsutum L.).

    PubMed

    Cheng, Wen-Han; Zhu, Hua-Guo; Tian, Wen-Gang; Zhu, Shou-Hong; Xiong, Xian-Peng; Sun, Yu-Qiang; Zhu, Qian-Hao; Sun, Jie

    2016-10-01

    Plant regeneration via somatic embryogenesis (SE) is the key step for genetic improvement of cotton (Gossypium hirsutum L.) through genetic engineering mediated by Agrobacteria, but the molecular mechanisms underlying SE in cotton is still unclear. Here, RNA-Sequencing was used to analyze the genes expressed during SE and their expression dynamics using RNAs isolated from non-embryogenic callus (NEC), embryogenic callus (EC) and somatic embryos (SEs). A total of 101, 670 unigenes were de novo assembled. The genes differentially expressed (DEGs) amongst NEC, EC and SEs were identified, annotated and classified. More DEGs were found between SEs and EC than between EC and NEC. A significant number of DEGs were related to hormone homeostasis, stress and ROS responses, and metabolism of polyamines. To confirm the expression dynamics of selected DEGs involved in various pathways, experiments were set up to investigate the effects of hormones (Indole-3-butytric acid, IBA; Kinetin, KT), polyamines, H2O2 and stresses on SE. Our results showed that exogenous application of IBA and KT positively regulated the development of EC and SEs, and that polyamines and H2O2 promoted the conversion of EC into SEs. Furthermore, we found that low and moderate stress is beneficial for proliferation of EC and SEs formation. Together, our global analysis of transcriptomic dynamics reveals that hormone homeostasis, polyamines, and stress response synergistically regulating SE in cotton. PMID:27511192

  6. Somatic embryogenesis and plant regeneration from immature zygotic embryos of papaya (Carica papaya L.).

    PubMed

    Fitch, M M; Manshardt, R M

    1990-10-01

    Immature zygotic embryos from open-pollinated and selfed Carica papaya L. fruits, 90 to 114 days post-anthesis, produced 2 to 20 somatic embryos on apical domes, cotyledonary nodes, and radicle meristems after culture for three weeks on half-strength Murashige and Skoog (MS) medium supplemented with 0.1 to 25 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), 400 mg l(-1) glutamine, and 6% sucrose. After six weeks of culture, about 40 to 50% of the zygotic embryos had become embryogenic, and each embryogenic embryo yielded hundreds of somatic embryos within five months of culture on media supplemented with 2,4-D. Somatic embryos matured on half-strength MS medium, germinated on MS medium containing 5 mg l(-1) kinetin, and grew large enough for greenhouse culture on MS medium. Shoots were rooted in vermiculite and grown in the greenhouse. PMID:24226942

  7. Plant regeneration via somatic embryogenesis in the seeded diploid banana Musa ornata Roxb.

    PubMed

    Cronauer-Mitra, S S; Krikorian, A D

    1988-01-01

    Somatic embryos of a seeded diploid ornamental banana (Musa ornata Roxb.) were obtained from zygotic embryos cultured on semi-solid Murashige and Skoog (MS) (1962) medium with the auxin 2,4-D (0.5, 1, 2 mg/l) and 5% CW. Removal of 2,4-D and transferral to Schenk and Hildebrandt (SH) (1972) salts with CW followed by basal MS led to embryo germination and growth. Plantlet production was obtained using filter paper bridges in liquid half-strength SH medium with 1% sucrose. The remarkable phenotypic fidelity of somatic embryos to that of zygotic embryos and the presence of a haustorium-like outgrowth on the somatic embryos is described. PMID:11538845

  8. 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

  9. An unusual abscisic acid and gibberellic acid synergism increases somatic embryogenesis, facilitates its genetic analysis and improves transformation in Medicago truncatula.

    PubMed

    Nolan, Kim E; Song, Youhong; Liao, Siyang; Saeed, Nasir A; Zhang, Xiyi; Rose, Ray J

    2014-01-01

    Somatic embryogenesis (SE) can be readily induced in leaf explants of the Jemalong 2HA genotype of the model legume Medicago truncatula by auxin and cytokinin, but rarely in wild-type Jemalong. Gibberellic acid (GA), a hormone not included in the medium, appears to act in Arabidopsis as a repressor of the embryonic state such that low ABA (abscisic acid): GA ratios will inhibit SE. It was important to evaluate the GA effect in M. truncatula in order to formulate generic SE mechanisms, given the Arabidopsis information. It was surprising to find that low ABA:GA ratios in M. truncatula acted synergistically to stimulate SE. The unusual synergism between GA and ABA in inducing SE has utility in improving SE for regeneration and transformation in M. truncatula. Expression of genes previously shown to be important in M. truncatula SE was not increased. In investigating genes previously studied in GA investigations of Arabidopsis SE, there was increased expression of GA2ox and decreased expression of PICKLE, a negative regulator of SE in Arabidopsis. We suggest that in M. truncatula there are different ABA:GA ratios required for down-regulating the PICKLE gene, a repressor of the embryonic state. In M. truncatula it is a low ABA:GA ratio while in Arabidopsis it is a high ABA:GA ratio. In different species the expression of key genes is probably related to differences in how the hormone networks optimise their expression. PMID:24937316

  10. 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

  11. 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

  12. Somatic embryogenesis, pigment accumulation, and synthetic seed production in Digitalis davisiana Heywood.

    PubMed

    Verma, Sandeep Kumar; Sahin, Gunce; Gurel, Ekrem

    2016-04-01

    Digitalis davisiana, commonly called Alanya foxglove, from Turkey, is an important medicinal herb as the main source of cardiac glycosides, cardenolides, anthraquinones, etc. It is also known in the Indian Medicine for treatment of wounds and burns. It has ornamental value as well. Overexploitation of D. davisiana has led this species to be declared protected, and thereby encouraged various methods for its propagation. In this study, an optimized and efficient plant tissue culture protocol was established using cotyledonary leaf, hypocotyl and root explants of D. davisiana. Callus tissues were obtained from the cotyledonary leaf, hypocotyl and root segments cultured on Murashige and Skoog's (MS) medium containing different plant growth regulators. The maximum number of somatic embryos were achieved by the MS medium containing 6-benzyladenine (1.0 mg/L BAP) or 2,4-dichlorophenoxy acetic acids (0.1 mg/L 2,4-D), which produced an average of 8.3 ± 1.5 or 5.3 ± 1.5 embryos per cotyledonary leaf, respectively. After 3 wk of culture in MS medium supplemented with 1.0 mg/L 2,4-D, callus showed a clear accumulation of orange pigmentation. Shoot regeneration was remarkably higher (14.3 indirect shoots) in a combination of α-naphthalene acetic acid (0.25 mg/L NAA) plus 3.0 mg/L BAP than 2.0 mg/L zeatin (10.3 ± 0.5 direct shoots) alone. The shoots were successfully rooted on MS medium supplemented with NAA (0.1-1.0 mg/L). In addition, synthetic seeds were produced by encapsulating shoot tips in 4% sodium alginate solution. Maximum conversion frequency of 76.6% was noted from encapsulated shoot tips cultured on 0.25 mg/L NAA with 1.0 mg/L BAP. The encapsulated shoot tips could be stored up to 60 days at 4 °C. Regenerated plantlets of D. davisiana were successfully acclimatized and transferred to soil. This study has demonstrated successful preservation of elite genotypes of D. davisiana. PMID:27295921

  13. 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

  14. 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

  15. The Conformation of a Plasma Membrane-Localized Somatic Embryogenesis Receptor Kinase Complex Is Altered by a Potato Aphid-Derived Effector.

    PubMed

    Peng, Hsuan-Chieh; Mantelin, Sophie; Hicks, Glenn R; Takken, Frank L W; Kaloshian, Isgouhi

    2016-07-01

    Somatic embryogenesis receptor kinases (SERKs) are transmembrane receptors involved in plant immunity. Tomato (Solanum lycopersicum) carries three SERK members. One of these, SlSERK1, is required for Mi-1.2-mediated resistance to potato aphids (Macrosiphum euphorbiae). Mi-1.2 encodes a coiled-coil nucleotide-binding leucine-rich repeat protein that in addition to potato aphids confers resistance to two additional phloem-feeding insects and to root-knot nematodes (Meloidogyne spp.). How SlSERK1 participates in Mi-1.2-mediated resistance is unknown, and no Mi-1.2 cognate pest effectors have been identified. Here, we study the mechanistic involvement of SlSERK1 in Mi-1.2-mediated resistance. We show that potato aphid saliva and protein extracts induce the Mi-1.2 defense marker gene SlWRKY72b, indicating that both saliva and extracts contain a Mi-1.2 recognized effector. Resistant tomato cultivar Motelle (Mi-1.2/Mi-1.2) plants overexpressing SlSERK1 were found to display enhanced resistance to potato aphids. Confocal microscopy revealed that Mi-1.2 localizes at three distinct subcellular compartments: the plasma membrane, cytoplasm, and nucleus. Coimmunoprecipitation experiments in these tomato plants and in Nicotiana benthamiana transiently expressing Mi-1.2 and SlSERK1 showed that Mi-1.2 and SlSERK1 colocalize only in a microsomal complex. Interestingly, bimolecular fluorescence complementation analysis showed that the interaction of Mi-1.2 and SlSERK1 at the plasma membrane distinctively changes in the presence of potato aphid saliva, suggesting a model in which a constitutive complex at the plasma membrane participates in defense signaling upon effector binding. PMID:27208261

  16. The Conformation of a Plasma Membrane-Localized Somatic Embryogenesis Receptor Kinase Complex Is Altered by a Potato Aphid-Derived Effector1[OPEN

    PubMed Central

    Peng, Hsuan-Chieh; Hicks, Glenn R.; Kaloshian, Isgouhi

    2016-01-01

    Somatic embryogenesis receptor kinases (SERKs) are transmembrane receptors involved in plant immunity. Tomato (Solanum lycopersicum) carries three SERK members. One of these, SlSERK1, is required for Mi-1.2-mediated resistance to potato aphids (Macrosiphum euphorbiae). Mi-1.2 encodes a coiled-coil nucleotide-binding leucine-rich repeat protein that in addition to potato aphids confers resistance to two additional phloem-feeding insects and to root-knot nematodes (Meloidogyne spp.). How SlSERK1 participates in Mi-1.2-mediated resistance is unknown, and no Mi-1.2 cognate pest effectors have been identified. Here, we study the mechanistic involvement of SlSERK1 in Mi-1.2-mediated resistance. We show that potato aphid saliva and protein extracts induce the Mi-1.2 defense marker gene SlWRKY72b, indicating that both saliva and extracts contain a Mi-1.2 recognized effector. Resistant tomato cultivar Motelle (Mi-1.2/Mi-1.2) plants overexpressing SlSERK1 were found to display enhanced resistance to potato aphids. Confocal microscopy revealed that Mi-1.2 localizes at three distinct subcellular compartments: the plasma membrane, cytoplasm, and nucleus. Coimmunoprecipitation experiments in these tomato plants and in Nicotiana benthamiana transiently expressing Mi-1.2 and SlSERK1 showed that Mi-1.2 and SlSERK1 colocalize only in a microsomal complex. Interestingly, bimolecular fluorescence complementation analysis showed that the interaction of Mi-1.2 and SlSERK1 at the plasma membrane distinctively changes in the presence of potato aphid saliva, suggesting a model in which a constitutive complex at the plasma membrane participates in defense signaling upon effector binding. PMID:27208261

  17. 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

  18. New insights into somatic embryogenesis: leafy cotyledon1, baby boom1 and WUSCHEL-related homeobox4 are epigenetically regulated in Coffea canephora.

    PubMed

    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.

  19. 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

    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

  20. Decreased GmAGL15 expression and reduced ethylene synthesis may contribute to reduced somatic embryogenesis in a poorly embryogenic cultivar of Glycine max.

    PubMed

    Zheng, Qiaolin; Zheng, Yumei; Perry, Sharyn E

    2013-09-01

    Somatic embryogenesis (SE) is the process by which cells become dedifferentiated and reprogram to follow an embryogenic pathway. It is important for regeneration of transgenic plants as well as for propagation of certain genotypes. However, competence for SE varies, even among genotypes of a species, and the basis for this variation is not understood. We have found that the MADS-box transcription factor (Glycine max) AGAMOUS-Like 15 [(Gm)AGL15] promotes SE in Arabidopsis and in soybean when overexpressed. In soybean, part of the promotion of SE is via GmAGL15-mediated control of ethylene biosynthesis and response. Addition of ACC, the precursor to ethylene, to culture media enhanced SE in Arabidopsis and soybean. Transcription factors important for embryogenesis responded directly to GmAGL15 and to ethylene accumulation. Here we correlate ethylene production and patterns of gene expression with SE potential of soybean genotypes. However, other results indicate that there is not a complete positive correlation between ethylene production and SE, indicating that the interactions between hormones, gene expression and developmental outcomes are complex.

  1. Decreased GmAGL15 expression and reduced ethylene synthesis may contribute to reduced somatic embryogenesis in a poorly embryogenic cultivar of Glycine max

    PubMed Central

    Zheng, Qiaolin; Zheng, Yumei; Perry, Sharyn E

    2013-01-01

    Somatic embryogenesis (SE) is the process by which cells become dedifferentiated and reprogram to follow an embryogenic pathway. It is important for regeneration of transgenic plants as well as for propagation of certain genotypes. However, competence for SE varies, even among genotypes of a species, and the basis for this variation is not understood. We have found that the MADS-box transcription factor (Glycine max) AGAMOUS-Like 15 [(Gm)AGL15] promotes SE in Arabidopsis and in soybean when overexpressed. In soybean, part of the promotion of SE is via GmAGL15-mediated control of ethylene biosynthesis and response. Addition of ACC, the precursor to ethylene, to culture media enhanced SE in Arabidopsis and soybean. Transcription factors important for embryogenesis responded directly to GmAGL15 and to ethylene accumulation. Here we correlate ethylene production and patterns of gene expression with SE potential of soybean genotypes. However, other results indicate that there is not a complete positive correlation between ethylene production and SE, indicating that the interactions between hormones, gene expression and developmental outcomes are complex. PMID:23838957

  2. 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

  3. Molecular evidence of true-to-type propagation of a 3-year-old Norway spruce through somatic embryogenesis.

    PubMed

    Harvengt, L; Trontin, J F; Reymond, I; Canlet, F; Paques, M

    2001-09-01

    Some progress has recently been made in establishing a system enabling somatic embryos to be initiated from old elite trees. We report here the first results demonstrating the molecular conformity of somatic embryos initiated from increasingly old Norway spruce (Picea abies (L.) Karst.), as indicated by an analysis of six nuclear microsatellites that showed an extremely high tendency to mutate during in vitro culture. No allelic difference was detected at these loci among plants regenerated from somatic embryos or between the former and mother plants. Moreover, phenotypical data acquired on the same 3- to 9-year-old plants growing in the field sampled for molecular analyses were totally in accord with the results on molecular conformity.

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. Expression of sex-specific molecular markers in clones of bipartite allophenic nemertines produced by somatic embryogenesis from Lineus sanguineus male/female chimera fragments.

    PubMed

    Tarpin, M; Bierne, J

    1995-04-01

    SDS-PAGE electrophoresis showed major sex-specific proteins in sexually maturing and mature Lineus sanguineus. These "egg-specific" (145, 78 and 40 kDa) and "sperm-specific" (55,52 and 28 kDa) proteins are useful for studying sex differentiation in bilaterally allophenic worms produced by asexual reproduction of bipartite male/female chimeric worms. This study was carried out on 2 symmetrical clones of bilaterally allophenic worms, derived by somatic embryogenesis from fragments transected from chimeras obtained by exchange-grafting lateral body halves of male and female specimens, and from their asexually-derived progeny. The electrophoretic patterns of proteins extracted from sexually immature, maturing and mature allophenic animals from the 5th to the 19th year of cloning, showed the presence of all female-specific markers and the absence of male-specific markers. There was also complete biochemical feminization of the male halves. The synthesis of the only egg-specific molecules in initially male lateral body halves means that the long-term cloning results in the total repression of genes encoding sperm-specific proteins, since genetically male determinant-bearing cells can randomly re-express the testis characteristic as fertile but rudimentary male gonads.

  9. 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, mi

  10. 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 e

  11. Transcriptional profiling of genes involved in embryogenic, non-embryogenic calluses and somatic embryogenesis of Valencia sweet orange by SSH-based microarray.

    PubMed

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

    2012-10-01

    Somatic embryogenesis (SE) is a most promising technology that is used for in vitro germplasm conservation and genetic improvement via biotechnological approaches in citrus. Herein, three suppression subtractive hybridization (SSH) libraries were constructed using calluses of Citrus sinensis cv. 'Valencia' to explore the molecular mechanisms that underlie the SE in citrus. A total of 880 unisequences were identified by microarray screening based on these three SSH libraries. Gene ontology analysis of the differentially expressed genes indicated that nucleolus associated regulation and biogenesis processes, hormone signal transduction, and stress factors might be involved in SE. Transcription factors might also play an important role. LEC1/B3 domain regulatory network genes (LEC1, L1L, FUS3, ABI3, and ABI5) were isolated in citrus SE. Some new transcription factors associated with citrus SE, like a B3 domain containing gene and HB4, were identified. To understand the influence of these isolated genes on SE competence, their expression profiles were compared among callus lines of seven citrus cultivars with different SE competence. The expression dynamics suggested that these genes could be necessary for the SE initiation and might play a role in embryogenic competence maintenance in different cultivars. On the basis of gene expression profiles, an overview of major physiological and biosynthesis processes at different developmental stages during citrus SE is presented. For the first time, these data provide a global resource for transcriptional events important for SE in citrus, and the specific genes offer new information for further investigation on citrus SE maintenance and development.

  12. 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.

  13. 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

  14. Advances in Reprogramming Somatic Cells to Induced Pluripotent Stem Cells

    PubMed Central

    Patel, Minal; Yang, Shuying

    2010-01-01

    Traditionally, nuclear reprogramming of cells has been performed by transferring somatic cell nuclei into oocytes, by combining somatic and pluripotent cells together through cell fusion and through genetic integration of factors through somatic cell chromatin. All of these techniques changes gene expression which further leads to a change in cell fate. Here we discuss recent advances in generating induced pluripotent stem cells, different reprogramming methods and clinical applications of iPS cells. Viral vectors have been used to transfer transcription factors (Oct4, Sox2, c-myc, Klf4, and nanog) to induce reprogramming of mouse fibroblasts, neural stem cells, neural progenitor cells, keratinocytes, B lymphocytes and meningeal membrane cells towards pluripotency. Human fibroblasts, neural cells, blood and keratinocytes have also been reprogrammed towards pluripotency. In this review we have discussed the use of viral vectors for reprogramming both animal and human stem cells. Currently, many studies are also involved in finding alternatives to using viral vectors carrying transcription factors for reprogramming cells. These include using plasmid transfection, piggyback transposon system and piggyback transposon system combined with a non viral vector system. Applications of these techniques have been discussed in detail including its advantages and disadvantages. Finally, current clinical applications of induced pluripotent stem cells and its limitations have also been reviewed. Thus, this review is a summary of current research advances in reprogramming cells into induced pluripotent stem cells. PMID:20336395

  15. Inflorescence proliferation for somatic embryogenesis induction and suspension-derived plant regeneration from banana (Musa AAA, cv. 'Dwarf Cavendish') male flowers.

    PubMed

    Pérez-Hernández, Juan Bernardo; Rosell-García, Purificación

    2008-06-01

    Availability of explants with adequate embryogenic competence is one of the most important limitations for the development of regenerable cell suspensions in banana. To increase the number and ease of accessibility to potentially embryogenic explants, a novel methodology is described by which young male flower clusters isolated from adult plants are induced to form new flower buds and proliferate in vitro. Different concentrations of the plant growth regulator thidiazuron (TDZ) induced inflorescence proliferation, which could be maintained over time as a continuous source of young flower buds. Intensity of proliferation was evaluated during successive subcultures. At the third cycle of proliferation, the highest multiplication rate (2.89) was obtained on the medium containing 5 microM TDZ. Newly generated floral tissues were assessed for embryogenic competence, resulting in an average embryogenic frequency of 12.5%. The observed embryogenic capacity, together with the recurrent availability of immature flowers, allowed for the direct initiation of cell suspensions from bulked explant cultures. Regular observation and regeneration tests during the development of suspended cell cultures confirmed their embryogenic condition. Produced embryos successfully matured and germinated to regenerate hundreds of somatic in vitro plants.

  16. Current methods for inducing pluripotency in somatic cells.

    PubMed

    Tavernier, Geertrui; Mlody, Barbara; Demeester, Jo; Adjaye, James; De Smedt, Stefaan C

    2013-05-28

    The groundbreaking discovery of reprogramming fibroblasts towards pluripotency merely by introducing four transcription factors (OCT4, SOX2, KLF4 and c-MYC) by means of retroviral transduction has created a promising revolution in the field of regenerative medicine. These so-called induced pluripotent stem cells (iPSCs) can provide a cell source for disease-modelling, drug-screening platforms, and transplantation strategies to treat incurable degenerative diseases, while circumventing the ethical issues and immune rejections associated with the use of non-autologous embryonic stem cells. The risk of insertional mutagenesis, caused both by the viral and transgene nature of the technique has proven to be the major limitation for iPSCs to be used in a clinical setting. In view of this, a variety of alternative techniques have been developed to induce pluripotency in somatic cells. This review provides an overview on current reprogramming protocols, discusses their pros and cons and future challenges to provide safe and transgene-free iPSCs.

  17. Induced DNA damage by dental resin monomers in somatic cells.

    PubMed

    Arossi, Guilherme Anziliero; Lehmann, Mauricio; Dihl, Rafael Rodrigues; Reguly, Maria Luiza; de Andrade, Heloisa Helena Rodrigues

    2010-02-01

    The present in vivo study investigated the genotoxicity of four dental resin monomers: triethyleneglycoldimethacrylate (TEGDMA), hydroxyethylmethacrylate (HEMA), urethanedimethacrylate (UDMA) and bisphenol A-glycidylmethacrylate (BisGMA). The Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster was applied to analyse their genotoxicity expressed as homologous mitotic recombination, point and chromosomal mutation. SMART detects the loss of heterozygosity of marker genes expressed phenotypically on the fly's wings. This fruit fly has an extensive genetic homology to mammalians, which makes it a suitable model organism for genotoxic investigations. The present findings provide evidence that the mechanistic basis underlying the genotoxicity of UDMA and TEGDMA is related to homologous recombination and gene/chromosomal mutation. A genotoxic pattern can correspondingly be discerned for both UDMA and TEGDMA: their genotoxicity is attributed respectively to 49% and 44% of mitotic recombination, as well as 51% and 56% of mutational events, including point and chromosomal alterations. The monomer UDMA is 1.6 times more active than TEGDMA to induce mutant clones per treatment unit. BisGMA and HEMA had no statistically significant effect on total spot frequencies - suggesting no genotoxic action in the SMART assay. The clinical significance of these observations has to be interpreted for data obtained in other bioassays.

  18. Aryl organophosphate flame retardants induced cardiotoxicity during zebrafish embryogenesis: by disturbing expression of the transcriptional regulators.

    PubMed

    Du, Zhongkun; Wang, Guowei; Gao, Shixiang; Wang, Zunyao

    2015-04-01

    As a result of the ban on some brominated flame retardants (BFRs), the use of organophosphate flame retardants (OPFRs) increases, and they are detected in multi-environment media at higher frequency and concentrations. However, the toxicity data of OPFRs, especially those on developmental toxicology are quite limited, which prevents an accurate evaluation of their environmental and health risk. Because a previous study reported that two aryl-OPFRs induced cardiotoxicity during zebrafish embryogenesis, we designed experiments to compare the heart developmental toxicity of a series of aryl-OPFRs with alkyl-OPFRs and explored possible internal mechanism. First, acute toxicity of 9 frequently used OPFRs were studied with zebrafish embryos (2-96 hpf). By comparing the LC50 and EC50 (pericardium edema) data, two aryl-OPFRs, triphenyl phosphate (TPhP) and cresyl diphenyl phosphate (CDP) showed greater heart developmental toxicity than the others. It was also found that the acute toxicity of OPFRs varied mainly depending on their hydrophobicity. Further study on the cardiotoxicity of TPhP and CDP showed that the cardiac looping progress can be impeded by 0.10mg/L TPhP or CDP exposure. Bradycardia and reduction of myocardium were also observed in 0.50 and 1.0mg/L TPhP groups and 0.10, 0.50, and 1.0mg/L CDP groups. 0-48 hpf is the vulnerable window of zebrafish cardiogenesis that can be easily affected by TPhP and CDP. RT-qPCR measurement on the expressions of key transcriptional regulators in cardiogenesis showed that BMP4, NKX2-5, and TBX5 were significantly inhibited at the exposure points of 12 hpf and 24 hpf which may be the internal factors related to the heart developmental toxicity. As zebrafish is a good model organism for human health study, the present results call for a greater attention to the health risk of fetus in pregnant women exposed to such OPFRs. PMID:25661707

  19. DNA methylation dynamics and MET1a-like gene expression changes during stress-induced pollen reprogramming to embryogenesis

    PubMed Central

    Testillano, Pilar S.

    2012-01-01

    Stress-induced plant cell reprogramming involves changes in global genome organization, being the epigenetic modifications key factors in the regulation of genome flexibility. DNA methylation, accomplished by DNA methyltransferases, constitutes a prominent epigenetic modification of the chromatin fibre which is locked in a transcriptionally inactive conformation. Changes in DNA methylation accompany the reorganization of the nuclear architecture during plant cell differentiation and proliferation. After a stress treatment, in vitro-cultured microspores are reprogrammed and change their gametophytic developmental pathway towards embryogenesis, the process constituting a useful system of reprogramming in isolated cells for applied and basic research. Gene expression driven by developmental and stress cues often depends on DNA methylation; however, global DNA methylation and genome-wide expression patterns relationship is still poorly understood. In this work, the dynamics of DNA methylation patterns in relation to nuclear architecture and the expression of BnMET1a-like DNA methyltransferase genes have been analysed during pollen development and pollen reprogramming to embryogenesis in Brassica napus L. by a multidisciplinary approach. Results showed an epigenetic reprogramming after microspore embryogenesis induction which involved a decrease of global DNA methylation and its nuclear redistribution with the change of developmental programme and the activation of cell proliferation, while DNA methylation increases with pollen and embryo differentiation in a cell-type-specific manner. Changes in the presence, abundance, and distribution of BnMET1a-like transcripts highly correlated with variations in DNA methylation. Mature zygotic and pollen embryos presented analogous patterns of DNA methylation and MET1a-like expression, providing new evidence of the similarities between both developmental embryogenic programmes. PMID:23175669

  20. 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 Dl

  1. 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

  2. Radiation-induced bystander signaling from somatic cells to germ cells in Caenorhabditis elegans.

    PubMed

    Guo, Xiaoying; Sun, Jie; Bian, Po; Chen, Lianyun; Zhan, Furu; Wang, Jun; Xu, An; Wang, Yugang; Hei, Tom K; Wu, Lijun

    2013-09-01

    Recently, radiation-induced bystander effects (RIBE) have been studied in mouse models in vivo, which clearly demonstrated bystander effects among somatic cells. However, there is currently no evidence for RIBE between somatic cells and germ cells in animal models in vivo. In the current study, the model animal Caenorhabditis elegans was used to investigate the bystander signaling from somatic cells to germ cells, as well as underlying mechanisms. C. elegans body size allows for precise microbeam irradiation and the abundant mutant strains for genetic dissection relative to currently adopted mouse models make it ideal for such analysis. Our results showed that irradiation of posterior pharynx bulbs and tails of C. elegans enhanced the level of germ cell apoptosis in bystander gonads. The irradiation of posterior pharynx bulbs also increased the level of DNA damage in bystander germ cells and genomic instability in the F1 progeny of irradiated worms, suggesting a potential carcinogenic risk in progeny even only somatic cells of parents are exposed to ionizing radiation (IR). It was also shown that DNA damage-induced germ cell death machinery and MAPK signaling pathways were both involved in the induction of germ cell apoptosis by microbeam induced bystander signaling, indicating a complex cooperation among multiple signaling pathways for bystander effects from somatic cells to germ cells.

  3. Chinmo is sufficient to induce male fate in somatic cells of the adult Drosophila ovary.

    PubMed

    Ma, Qing; de Cuevas, Margaret; Matunis, Erika L

    2016-03-01

    Sexual identity is continuously maintained in specific differentiated cell types long after sex determination occurs during development. In the adult Drosophila testis, the putative transcription factor Chronologically inappropriate morphogenesis (Chinmo) acts with the canonical male sex determinant DoublesexM (Dsx(M)) to maintain the male identity of somatic cyst stem cells and their progeny. Here we find that ectopic expression of chinmo is sufficient to induce a male identity in adult ovarian somatic cells, but it acts through a Dsx(M)-independent mechanism. Conversely, the feminization of the testis somatic stem cell lineage caused by loss of chinmo is enhanced by expression of the canonical female sex determinant Dsx(F), indicating that chinmo acts in parallel with the canonical sex determination pathway to maintain the male identity of testis somatic cells. Consistent with this finding, ectopic expression of female sex determinants in the adult testis disrupts tissue morphology. The miRNA let-7 downregulates chinmo in many contexts, and ectopic expression of let-7 in the adult testis is sufficient to recapitulate the chinmo loss-of-function phenotype, but we find no apparent phenotypes upon removal of let-7 in the adult ovary or testis. Our finding that chinmo is necessary and sufficient to promote a male identity in adult gonadal somatic cells suggests that the sexual identity of somatic cells can be reprogrammed in the adult Drosophila ovary as well as in the testis. PMID:26811385

  4. Mutational History of a Human Cell Lineage from Somatic to Induced Pluripotent Stem Cells

    PubMed Central

    Rouhani, Foad J.; Nik-Zainal, Serena; Wuster, Arthur; Li, Yilong; Conte, Nathalie; Koike-Yusa, Hiroko; Kumasaka, Natsuhiko; Vallier, Ludovic; Yusa, Kosuke; Bradley, Allan

    2016-01-01

    The accuracy of replicating the genetic code is fundamental. DNA repair mechanisms protect the fidelity of the genome ensuring a low error rate between generations. This sustains the similarity of individuals whilst providing a repertoire of variants for evolution. The mutation rate in the human genome has recently been measured to be 50–70 de novo single nucleotide variants (SNVs) between generations. During development mutations accumulate in somatic cells so that an organism is a mosaic. However, variation within a tissue and between tissues has not been analysed. By reprogramming somatic cells into induced pluripotent stem cells (iPSCs), their genomes and the associated mutational history are captured. By sequencing the genomes of polyclonal and monoclonal somatic cells and derived iPSCs we have determined the mutation rates and show how the patterns change from a somatic lineage in vivo through to iPSCs. Somatic cells have a mutation rate of 14 SNVs per cell per generation while iPSCs exhibited a ten-fold lower rate. Analyses of mutational signatures suggested that deamination of methylated cytosine may be the major mutagenic source in vivo, whilst oxidative DNA damage becomes dominant in vitro. Our results provide insights for better understanding of mutational processes and lineage relationships between human somatic cells. Furthermore it provides a foundation for interpretation of elevated mutation rates and patterns in cancer. PMID:27054363

  5. 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.

  6. Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells.

    PubMed

    Abyzov, Alexej; Mariani, Jessica; Palejev, Dean; Zhang, Ying; Haney, Michael Seamus; Tomasini, Livia; Ferrandino, Anthony F; Rosenberg Belmaker, Lior A; Szekely, Anna; Wilson, Michael; Kocabas, Arif; Calixto, Nathaniel E; Grigorenko, Elena L; Huttner, Anita; Chawarska, Katarzyna; Weissman, Sherman; Urban, Alexander Eckehart; Gerstein, Mark; Vaccarino, Flora M

    2012-12-20

    Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) has been suspected of causing de novo copy number variation. To explore this issue, here we perform a whole-genome and transcriptome analysis of 20 human iPSC lines derived from the primary skin fibroblasts of seven individuals using next-generation sequencing. We find that, on average, an iPSC line manifests two copy number variants (CNVs) not apparent in the fibroblasts from which the iPSC was derived. Using PCR and digital droplet PCR, we show that at least 50% of those CNVs are present as low-frequency somatic genomic variants in parental fibroblasts (that is, the fibroblasts from which each corresponding human iPSC line is derived), and are manifested in iPSC lines owing to their clonal origin. Hence, reprogramming does not necessarily lead to de novo CNVs in iPSCs, because most of the line-manifested CNVs reflect somatic mosaicism in the human skin. Moreover, our findings demonstrate that clonal expansion, and iPSC lines in particular, can be used as a discovery tool to reliably detect low-frequency CNVs in the tissue of origin. Overall, we estimate that approximately 30% of the fibroblast cells have somatic CNVs in their genomes, suggesting widespread somatic mosaicism in the human body. Our study paves the way to understanding the fundamental question of the extent to which cells of the human body normally acquire structural alterations in their DNA post-zygotically.

  7. Hexavalent chromium induces apoptosis in male somatic and spermatogonial stem cells via redox imbalance

    PubMed Central

    Das, Joydeep; Kang, Min-Hee; Kim, Eunsu; Kwon, Deug-Nam; Choi, Yun-Jung; Kim, Jin-Hoi

    2015-01-01

    Hexavalent chromium [Cr(VI)], an environmental toxicant, causes severe male reproductive abnormalities. However, the actual mechanisms of toxicity are not clearly understood and have not been studied in detail. The present in vitro study aimed to investigate the mechanism of reproductive toxicity of Cr(VI) in male somatic cells (mouse TM3 Leydig cells and TM4 Sertoli cells) and spermatogonial stem cells (SSCs) because damage to or dysfunction of these cells can directly affect spermatogenesis, resulting in male infertility. Cr(VI) by inducing oxidative stress was cytotoxic to both male somatic cells and SSCs in a dose-dependent manner, and induced mitochondria-dependent apoptosis. Although the mechanism of Cr(VI)-induced cytotoxicity was similar in both somatic cells, the differences in sensitivity of TM3 and TM4 cells to Cr(VI) could be attributed, at least in part, to cell-specific regulation of P-AKT1, P-ERK1/2, and P-P53 proteins. Cr(VI) affected the differentiation and self-renewal mechanisms of SSCs, disrupted steroidogenesis in TM3 cells, while in TM4 cells, the expression of tight junction signaling and cell receptor molecules was affected as well as the secretory functions were impaired. In conclusion, our results show that Cr(VI) is cytotoxic and impairs the physiological functions of male somatic cells and SSCs. PMID:26355036

  8. Enhanced homologous recombination is induced by alpha-particle radiation in somatic cells of Arabidopsis thaliana

    NASA Astrophysics Data System (ADS)

    Bian, Po; Liu, Ping; Wu, Yuejin

    Almost 9 percent of cosmic rays which strike the earth's atmosphere are alpha particles. As one of the ionizing radiations (IR), its biological effects have been widely studied. However, the plant genomic instability induced by alpha-particle radiation was not largely known. In this research, the Arabidopsis thaliana transgenic for GUS recombination substrate was used to evaluate the genomic instability induced by alpha-particle radiation (3.3MeV). The pronounced effects of systemic exposure to alpha-particle radiation on the somatic homologous recombination frequency (HRF) were found at different doses. The 10Gy dose of radiation induced the maximal HRF which was 1.9-fold higher than the control. The local radiation of alpha-particle (10Gy) on root also resulted in a 2.5-fold increase of somatic HRF in non-radiated aerial plant, indicating that the signal(s) of genomic instability was transferred to non-radiated parts and initiated their genomic instability. Concurrent treatment of seedlings of Arabidopsis thaliana with alpha-particle and DMSO(ROS scavenger) both in systemic and local radiation signifi- cantly suppressed the somatic HR, indicating that the free radicals produced by alpha-particle radiation took part in the production of signal of genomic instability rather than the signal transfer. Key words: alpha-particle radiation, somatic homologous recombination, genomic instability

  9. 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

  10. 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.

  11. [Mammalian DNA methylation and its roles during the induced re-programming of somatic cells].

    PubMed

    Hongwei, Song; Tiezhu, An; Shanhua, Piao; Chunsheng, Wang

    2014-05-01

    The technology of induced pluripotent stem cell (iPS) provides the possibility to reverse the terminal differentiated cells to pluripotent stem cells, and is therefore of great importance in both the theoretical research of stem cells and regenerative medicine. However, the efficiency of current induced reprogramming methods is extremely low, and the incomplete reprogramming often happens. It has been reported that some epigenetic memory of the somatic cells exists in these incomplete reprogrammed iPS cells, and DNA methylation, as a relative long-term and stable epigenetic modification, is one of the important factors that influence the efficiency of reprogramming and differentiative capacity of iPS cells. Mammalian DNA methylation, which normally appears on the CpG sites, occurs on the fifth carbon atom of the cytosine ring. DNA methylation can modulate the expression of somatic cell specific genes, and pluripotent genes; hence, it plays important roles in the processes of mammalian gene regulation, embryonic development and cell reprogramming. In addition, it has also been found that abnormal DNA methylation may lead to the disorder of genetic imprinting and the inactivation of X chromosome in iPS cells. Therefore, in order to provide a concise guidance of DNA methylation studies in iPS, we mainly review the mechanism, the distribution features of DNA methylation, and its roles in induced reprogramming of somatic cells. PMID:24846992

  12. Moderate malnutrition in rats induces somatic gene mutations.

    PubMed

    Pacheco-Martínez, M Monserrat; Cortés-Barberena, Edith; Cervantes-Ríos, Elsa; Del Carmen García-Rodríguez, María; Rodríguez-Cruz, Leonor; Ortiz-Muñiz, Rocío

    2016-07-01

    The relationship between malnutrition and genetic damage has been widely studied in human and animal models, leading to the observation that interactions between genotoxic exposure and micronutrient status appear to affect genomic stability. A new assay has been developed that uses the phosphatidylinositol glycan class A gene (Pig-a) as a reporter for measuring in vivo gene mutation. The Pig-a assay can be employed to evaluate mutant frequencies (MFs) in peripheral blood reticulocytes (RETs) and erythrocytes (RBCs) using flow cytometry. In the present study, we assessed the effects of malnutrition on mutagenic susceptibility by exposing undernourished (UN) and well-nourished (WN) rats to N-ethyl-N-nitrosourea (ENU) and measuring Pig-a MFs. Two week-old UN and WN male Han-Wistar rats were treated daily with 0, 20, or 40mg/kg ENU for 3 consecutive days. Blood was collected from the tail vein one day before ENU treatment (Day-1) and after ENU administration on Days 7, 14, 21, 28, 35, 42, 49, 56 and 63. Pig-a MFs were measured in RETs and RBCs as the RET(CD59-) and RBC(CD59-) frequencies. In the vehicle control groups, the frequencies of mutant RETs and RBCs were significantly higher in UN rats compared with WN rats at all sampling times. The ENU treatments increased RET and RBC MFs starting at Day 7. Although ENU-induced Pig-a MFs were consistently lower in UN rats than in WN rats, these differences were not significant. To understand these responses, further studies should use other mutagens and nucleated surrogate cells and examine the types of mutations induced in UN and WN rats.

  13. Moderate malnutrition in rats induces somatic gene mutations.

    PubMed

    Pacheco-Martínez, M Monserrat; Cortés-Barberena, Edith; Cervantes-Ríos, Elsa; Del Carmen García-Rodríguez, María; Rodríguez-Cruz, Leonor; Ortiz-Muñiz, Rocío

    2016-07-01

    The relationship between malnutrition and genetic damage has been widely studied in human and animal models, leading to the observation that interactions between genotoxic exposure and micronutrient status appear to affect genomic stability. A new assay has been developed that uses the phosphatidylinositol glycan class A gene (Pig-a) as a reporter for measuring in vivo gene mutation. The Pig-a assay can be employed to evaluate mutant frequencies (MFs) in peripheral blood reticulocytes (RETs) and erythrocytes (RBCs) using flow cytometry. In the present study, we assessed the effects of malnutrition on mutagenic susceptibility by exposing undernourished (UN) and well-nourished (WN) rats to N-ethyl-N-nitrosourea (ENU) and measuring Pig-a MFs. Two week-old UN and WN male Han-Wistar rats were treated daily with 0, 20, or 40mg/kg ENU for 3 consecutive days. Blood was collected from the tail vein one day before ENU treatment (Day-1) and after ENU administration on Days 7, 14, 21, 28, 35, 42, 49, 56 and 63. Pig-a MFs were measured in RETs and RBCs as the RET(CD59-) and RBC(CD59-) frequencies. In the vehicle control groups, the frequencies of mutant RETs and RBCs were significantly higher in UN rats compared with WN rats at all sampling times. The ENU treatments increased RET and RBC MFs starting at Day 7. Although ENU-induced Pig-a MFs were consistently lower in UN rats than in WN rats, these differences were not significant. To understand these responses, further studies should use other mutagens and nucleated surrogate cells and examine the types of mutations induced in UN and WN rats. PMID:26994962

  14. 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

  15. Developmentally and stress-induced small heat shock proteins in cork oak somatic embryos.

    PubMed

    Puigderrajols, Pere; Jofré, Anna; Mir, Gisela; Pla, Maria; Verdaguer, Dolors; Huguet, Gemma; Molinas, Marisa

    2002-06-01

    The timing and tissue localization of small heat shock proteins (sHSPs) during cork oak somatic embryo development was investigated under normal growing culture conditions and in response to stress. Western blot analyses using polyclonal antibodies raised against cork oak recombinant HSP17 showed a transient accumulation of class I sHSPs during somatic embryo maturation and germination. Moreover, the amount of protein increased at all stages of embryo development in response to exogenous stress. The developmentally accumulated proteins localized to early differentiating, but not the highly dividing, regions of the root and shoot apical meristems. By contrast, these highly dividing regions were strongly immunostained after heat stress. Findings support the hypothesis of a distinct control for developmentally and stress-induced accumulation of class I sHSPs. The possible role of sHSPs is discussed in relation to their tissue specific localization.

  16. Developmental regulation of neuroligin genes in Japanese ricefish (Oryzias latipes) embryogenesis maintains the rhythm during ethanol-induced fetal alcohol spectrum disorder.

    PubMed

    Haron, Mona H; Khan, Ikhlas A; Dasmahapatra, Asok K

    2014-01-01

    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 ricefish (Oryzias latipes) embryogenesis as an animal model of FASD and reported that this model has effectively generated several phenotypic features in the cardiovasculature and neurocranial cartilages by developmental ethanol exposure which is analogous to human FASD phenotypes. As FASD is a neurobehavioral disorder, we are searching for a molecular target of ethanol that alters neurological functions. In this communication, we have focused on neuroligin genes (nlgn) which are known to be active at the postsynaptic side of both excitatory and inhibitory synapses of the central nervous system. There are six human NLGN homologs of Japanese ricefish reported in public data bases. We have partially cloned these genes and analyzed their expression pattern during normal development and also after exposing the embryos to ethanol. Our data indicate that the expression of all six nlgn genes in Japanese ricefish embryos is developmentally regulated. Although ethanol is able to induce developmental abnormalities in Japanese ricefish embryogenesis comparable to the FASD phenotypes, quantitative real-time PCR (qPCR) analysis of nlgn mRNAs indicate unresponsiveness of these genes to ethanol. We conclude that the disruption of the developmental rhythm of Japanese ricefish embryogenesis by ethanol that leads to FASD may not affect the nlgn gene expression at the message level.

  17. Genotoxicity of neutrons in Drosophila melanogaster. Somatic mutation and recombination induced by reactor neutrons.

    PubMed

    Guzmán-Rincón, J; Delfín-Loya, A; Ureña-Núñez, F; Paredes, L C; Zambrano-Achirica, F; Graf, U

    2005-08-01

    This paper describes the observation of a direct relationship between the absorbed doses of neutrons and the frequencies of somatic mutation and recombination using the wing somatic mutation and recombination test (SMART) of Drosophila melanogaster. This test was used for evaluating the biological effects induced by neutrons from the Triga Mark III reactor of Mexico. Two different reactor power levels were used, 300 and 1000 kW, and two absorbed doses were tested for each power level: 1.6 and 3.2 Gy for 300 kW and 0.84 and 1.7 Gy for 1000 kW. A linear relationship was observed between the absorbed dose and the somatic mutation and recombination frequencies. Furthermore, these frequencies were dependent on larval age: In 96-h-old larvae, the frequencies were increased considerably but the sizes of the spots were smaller than in 72-h-old larvae. The analysis of the balancer-heterozygous progeny showed a linear absorbed dose- response relationship, although the responses were clearly lower than found in the marker-trans-heterozygous flies. Approximately 65% of the genotoxicity observed is due to recombinational events. The results of the study indicate that thermal and fast neutrons are both mutagenic and recombinagenic in the D. melanogaster wing SMART, and that the frequencies are dependent on neutron dose, reactor power, and the age of the treated larvae.

  18. Genotoxicity of neutrons in Drosophila melanogaster. Somatic mutation and recombination induced by reactor neutrons.

    PubMed

    Guzmán-Rincón, J; Delfín-Loya, A; Ureña-Núñez, F; Paredes, L C; Zambrano-Achirica, F; Graf, U

    2005-08-01

    This paper describes the observation of a direct relationship between the absorbed doses of neutrons and the frequencies of somatic mutation and recombination using the wing somatic mutation and recombination test (SMART) of Drosophila melanogaster. This test was used for evaluating the biological effects induced by neutrons from the Triga Mark III reactor of Mexico. Two different reactor power levels were used, 300 and 1000 kW, and two absorbed doses were tested for each power level: 1.6 and 3.2 Gy for 300 kW and 0.84 and 1.7 Gy for 1000 kW. A linear relationship was observed between the absorbed dose and the somatic mutation and recombination frequencies. Furthermore, these frequencies were dependent on larval age: In 96-h-old larvae, the frequencies were increased considerably but the sizes of the spots were smaller than in 72-h-old larvae. The analysis of the balancer-heterozygous progeny showed a linear absorbed dose- response relationship, although the responses were clearly lower than found in the marker-trans-heterozygous flies. Approximately 65% of the genotoxicity observed is due to recombinational events. The results of the study indicate that thermal and fast neutrons are both mutagenic and recombinagenic in the D. melanogaster wing SMART, and that the frequencies are dependent on neutron dose, reactor power, and the age of the treated larvae. PMID:16038586

  19. Limiting replication stress during somatic cell reprogramming reduces genomic instability in induced pluripotent stem cells

    PubMed Central

    Ruiz, Sergio; Lopez-Contreras, Andres J.; Gabut, Mathieu; Marion, Rosa M.; Gutierrez-Martinez, Paula; Bua, Sabela; Ramirez, Oscar; Olalde, Iñigo; Rodrigo-Perez, Sara; Li, Han; Marques-Bonet, Tomas; Serrano, Manuel; Blasco, Maria A.; Batada, Nizar N.; Fernandez-Capetillo, Oscar

    2015-01-01

    The generation of induced pluripotent stem cells (iPSC) from adult somatic cells is one of the most remarkable discoveries in recent decades. However, several works have reported evidence of genomic instability in iPSC, raising concerns on their biomedical use. The reasons behind the genomic instability observed in iPSC remain mostly unknown. Here we show that, similar to the phenomenon of oncogene-induced replication stress, the expression of reprogramming factors induces replication stress. Increasing the levels of the checkpoint kinase 1 (CHK1) reduces reprogramming-induced replication stress and increases the efficiency of iPSC generation. Similarly, nucleoside supplementation during reprogramming reduces the load of DNA damage and genomic rearrangements on iPSC. Our data reveal that lowering replication stress during reprogramming, genetically or chemically, provides a simple strategy to reduce genomic instability on mouse and human iPSC. PMID:26292731

  20. MicroRNA-mediated somatic cell reprogramming.

    PubMed

    Kuo, Chih-Hao; Ying, Shao-Yao

    2013-02-01

    Since the first report of induced pluripotent stem cells (iPSCs) using somatic cell nuclear transfer (SCNT), much focus has been placed on iPSCs due to their great therapeutic potential for diseases such as abnormal development, degenerative disorders, and even cancers. Subsequently, Takahashi and Yamanaka took a novel approach by using four defined transcription factors to generate iPSCs in mice and human fibroblast cells. Scientists have since been trying to refine or develop better approaches to reprogramming, either by using different combinations of transcription factors or delivery methods. However, recent reports showed that the microRNA expression pattern plays a crucial role in somatic cell reprogramming and ectopic introduction of embryonic stem cell-specific microRNAs revert cells back to an ESC-like state, although, the exact mechanism underlying this effect remains unclear. This review describes recent work that has focused on microRNA-mediated approaches to somatic cell reprogramming as well as some of the pros and cons to these approaches and a possible mechanism of action. Based on the pivotal role of microRNAs in embryogenesis and somatic cell reprogramming, studies in this area must continue in order to gain a better understanding of the role of microRNAs in stem cells regulation and activity. PMID:22961769

  1. Superior efficacy of secreted over somatic antigen display in recombinant Salmonella vaccine induced protection against listeriosis.

    PubMed Central

    Hess, J; Gentschev, I; Miko, D; Welzel, M; Ladel, C; Goebel, W; Kaufmann, S H

    1996-01-01

    Vaccination provides the most potent measure against infectious disease, and recombinant (r) viable vaccines expressing defined pathogen-derived antigens represent powerful candidates for future vaccination strategies. In a new approach we constructed r-aroA- Salmonella typhimurium displaying p60 or listeriolysin (Hly) antigen of Listeria monocytogenes in secreted or somatic form in the host cell. Vaccination of mice with r-aroA- S. typhimurium induced protection against the intracellular pathogen L. monocytogenes only with secreted and not with somatic antigen. Secreted Hly was slightly more potent in inducing protective immunity than secreted p60. Both r-aroA- S. typhimurium secreting p60 in the endosome and r-aroA- S. typhimurium secreting Hly in the cytosol induced protective CD4+ and CD8+ T-cells suggesting CD8+ T-cell stimulation independent from intracellular residence of r-aroA- S. typhimurium carriers. Hence, not only the type of antigen but also its display by the r-carrier within the host cell critically influences vaccine efficacy. Images Fig. 1 PMID:8643654

  2. Inducing somatic meiosis-like reduction at high frequency by caffeine in root-tip cells of Vicia faba.

    PubMed

    Chen, Y; Zhang, L; Zhou, Y; Geng, Y; Chen, Z

    2000-07-20

    Germinated seeds of Vicia faba were treated in caffeine solutions of different concentration for different durations to establish the inducing system of somatic meiosis-like reduction. The highest frequency of somatic meiosis-like reduction could reach up to 54.0% by treating the root tips in 70 mmol/l caffeine solution for 2 h and restoring for 24 h. Two types of somatic meiosis-like reduction were observed. One was reductional grouping, in which the chromosomes in a cell usually separated into two groups, and the role of spindle fibers did not show. The other type was somatic meiosis, which was analogous to meiosis presenting in gametogenesis, and chromosome pairing and chiasmata were visualized.

  3. Spontaneous and induced chromosome damage in somatic cells of sporadic and familial Alzheimer's disease patients.

    PubMed

    Trippi, F; Botto, N; Scarpato, R; Petrozzi, L; Bonuccelli, U; Latorraca, S; Sorbi, S; Migliore, L

    2001-07-01

    Alzheimer's disease (AD) is a neurodegenerative disorder of the elderly with a complex etiology due to the interaction between genetic and environmental factors. At least 15% of cases are inherited as an autosomal dominant mutation, but the majority are sporadic. We evaluated cytogenetic alterations, both spontaneous and chemical-induced [aluminium (Al) and griseofulvin (GF)], by means of the micronucleus (MN) test in lymphocytes or skin fibroblasts of 14 patients with sporadic and eight with familial Alzheimer's disease (FAD), respectively. The spontaneous MN frequencies of sporadic (20.8 +/- 9.2) and familial (20.7 +/- 4.6) AD patients are significantly higher than those of the respective control groups (9.0 +/- 6.8 and 6.7 +/- 3.4). In all AD patients, GF significantly increased the spontaneous MN frequency of somatic cells to a lesser extent (P < 0.05) as compared with the control group. Al treatment did not induce MN in AD patients. The results of the present study indicate that different types of somatic cells from sporadic and familial AD patients show comparable levels of spontaneous cytogenetic anomalies, and MN induction is partially reduced or lacking according to the type of chemical treatments. PMID:11420400

  4. Placebo-Induced Somatic Sensations: A Multi-Modal Study of Three Different Placebo Interventions

    PubMed Central

    Beissner, Florian; Brünner, Franziska; Fink, Maria; Meissner, Karin; Kaptchuk, Ted J.; Napadow, Vitaly

    2015-01-01

    Somatic sensations induced by placebos are a frequent phenomenon whose etiology and clinical relevance remains unknown. In this study, we have evaluated the quantitative, qualitative, spatial, and temporal characteristics of placebo-induced somatic sensations in response to three different placebo interventions: (1) placebo irritant solution, (2) placebo laser stimulation, and (3) imagined laser stimulation. The quality and intensity of evoked sensations were assessed using the McGill pain questionnaire and visual analogue scales (VAS), while subjects’ sensation drawings processed by a geographic information system (GIS) were used to measure their spatial characteristics. We found that all three interventions are capable of producing robust sensations most frequently described as “tingling” and “warm” that can reach consider-able spatial extent (≤ 205mm²) and intensity (≤ 80/100 VAS). Sensations from placebo stimulation were often referred to areas remote from the stimulation site and exhibit considerable similarity with referred pain. Interestingly, there was considerable similarity of qualitative features as well as spatial patterns across subjects and placebos. However, placebo laser stimulation elicited significantly stronger and more widespread sensations than placebo irritant solution. Finally, novelty seeking, a character trait assessed by the Temperament and Character Inventory and associated with basal dopaminergic activity, was less pronounced in subjects susceptible to report placebo-induced sensations. Our study has shown that placebo-induced sensations are frequent and can reach considerable intensity and extent. As multiple somatosensory subsystems are involved despite the lack of peripheral stimulus, we propose a central etiology for this phenomenon. PMID:25901350

  5. A change of developmental program induces the remodeling of the interchromatin domain during microspore embryogenesis in Brassica napus L.

    PubMed

    Seguí-Simarro, J M; Corral-Martínez, P; Corredor, E; Raska, I; Testillano, P S; Risueño, M C

    2011-05-15

    After a stress treatment, in vitro-cultured pollen changes its normal gametophytic developmental pathway towards embryogenesis producing multicellular embryos from which, finally, haploid and double haploid plants develop. The architecture of the well-organized nuclear functional domains changes in response to DNA replication, RNA transcription, processing and transport dynamics. A number of subnuclear structures present in the interchromatin region (IR, the nuclear domain between chromosome territories) have been shown as involved, either directly or indirectly, in transcriptional regulation. These structures include the interchromatin granule clusters (IGCs), perichromatin fibrils (PFs), Cajal bodies (CBs) and perichromatin granules (PGs). In this work, we present a cytochemical, immunocytochemical, quantitative and morphometric analysis at the light, confocal and electron microscopy levels to characterize the changes in the functional architecture of the nuclear interchromatin domain during two developmental programs followed by the microspore: differentiation to mature pollen grains (transcriptionally inactive), and microspore embryogenesis involving proliferation in the first stages (highly engaged in transcription). Our results revealed characteristic changes in size, shape and distribution of the different interchromatin structures as a consequence of the reprogramming of the microspore, allowing us to relate the remodeling of the interchromatin domain to the variations in transcriptional activities during proliferation and differentiation events, and suggesting that RNA-associated structures could be a regulatory mechanism in the process. In addition, we document the presence of two structurally different types of CBs, and of IGC and CB-associated regions, similar to those present in animal cells, and not yet described in plants.

  6. Chemically Induced Reprogramming of Somatic Cells to Pluripotent Stem Cells and Neural Cells

    PubMed Central

    Biswas, Dhruba; Jiang, Peng

    2016-01-01

    The ability to generate transplantable neural cells in a large quantity in the laboratory is a critical step in the field of developing stem cell regenerative medicine for neural repair. During the last few years, groundbreaking studies have shown that cell fate of adult somatic cells can be reprogrammed through lineage specific expression of transcription factors (TFs)-and defined culture conditions. This key concept has been used to identify a number of potent small molecules that could enhance the efficiency of reprogramming with TFs. Recently, a growing number of studies have shown that small molecules targeting specific epigenetic and signaling pathways can replace all of the reprogramming TFs. Here, we provide a detailed review of the studies reporting the generation of chemically induced pluripotent stem cells (ciPSCs), neural stem cells (ciNSCs), and neurons (ciN). We also discuss the main mechanisms of actions and the pathways that the small molecules regulate during chemical reprogramming. PMID:26861316

  7. Cell therapy using induced pluripotent stem cells or somatic stem cells: this is the question.

    PubMed

    Somoza, Rodrigo A; Rubio, Francisco J

    2012-05-01

    A lot of effort has been developed to bypass the use of embryonic stem cells (ES) in human therapies, because of several concerns and ethical issues. Some unsolved problems of using stem cells for human therapies, excluding the human embryonic origin, are: how to regulate cell plasticity and proliferation, immunological compatibility, potential adverse side-effects when stem cells are systemically administrated, and the in vivo signals to rule out a specific cell fate after transplantation. Currently, it is known that almost all tissues of an adult organism have somatic stem cells (SSC). Whereas ES are primary involved in the genesis of new tissues and organs, SSC are involved in regeneration processes, immuno-regulatory and homeostasis mechanisms. Although the differentiating potential of ES is higher than SSC, several studies suggest that some types of SSC, such as mesenchymal stem cells (MSC), can be induced epigenetically to differentiate into tissue-specific cells of different lineages. This unexpected pluripotency and the variety of sources that they come from, can make MSC-like cells suitable for the treatment of diverse pathologies and injuries. New hopes for cell therapy came from somatic/mature cells and the discovery that could be reprogrammed to a pluripotent stage similar to ES, thus generating induced pluripotent stem cells (iPS). For this, it is necessary to overexpress four main reprogramming factors, Sox2, Oct4, Klf4 and c-Myc. The aim of this review is to analyze the potential and requirements of cellular based tools in human therapy strategies, focusing on the advantage of using MSC over iPS.

  8. 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.

  9. Effects of tannic acid on spontaneous and induced somatic mutations in Drosophila melanogaster.

    PubMed

    Szakmary, A; Knasmüller, S

    1991-05-01

    The effects of tannic acid (TA) alone and in combination with direct acting chemical genotoxins and gamma-radiation were investigated in Somatic Mutation and Recombination Tests (SMARTs) using Drosophila melanogaster. Treatment with TA alone (2.5-15 mmol/l) resulted in a moderate but dose dependent induction of mosaic spots in males and females, indicating that the compound possesses mutagenic and recombinogenic activity. When TA (10 mmol/l) was given simultaneously with MMS, 4-NQO and cis-DDP, a potentiating effect on their mutagenicity was observed in males whereas in females no such increase was measured. The frequency of 4-NQO induced mosaic spots in males was raised more than threefold in presence of TA, for MMS and cis-DDP the enhancement was approximately 2-fold; with gamma-radiation no synergistic effect occurred. The different response in the two sexes indicates that TA preferentially induces gene mutations and deletions but has no enhancing effect on the number of mosaic spots which are formed as a consequence of recombinogenic events. PMID:1908938

  10. 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

  11. 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.

  12. A late embryogenesis abundant protein HVA1 regulated by an inducible promoter enhances root growth and abiotic stress tolerance in rice without yield penalty.

    PubMed

    Chen, Yi-Shih; Lo, Shuen-Fang; Sun, Peng-Kai; Lu, Chung-An; Ho, Tuan-Hua D; Yu, Su-May

    2015-01-01

    Regulation of root architecture is essential for maintaining plant growth under adverse environment. A synthetic abscisic acid (ABA)/stress-inducible promoter was designed to control the expression of a late embryogenesis abundant protein (HVA1) in transgenic rice. The background of HVA1 is low but highly inducible by ABA, salt, dehydration and cold. HVA1 was highly accumulated in root apical meristem (RAM) and lateral root primordia (LRP) after ABA/stress treatments, leading to enhanced root system expansion. Water-use efficiency (WUE) and biomass also increased in transgenic rice, likely due to the maintenance of normal cell functions and metabolic activities conferred by HVA1 which is capable of stabilizing proteins, under osmotic stress. HVA1 promotes lateral root (LR) initiation, elongation and emergence and primary root (PR) elongation via an auxin-dependent process, particularly by intensifying asymmetrical accumulation of auxin in LRP founder cells and RAM, even under ABA/stress-suppressive conditions. We demonstrate a successful application of an inducible promoter in regulating the spatial and temporal expression of HVA1 for improving root architecture and multiple stress tolerance without yield penalty. PMID:25200982

  13. A late embryogenesis abundant protein HVA1 regulated by an inducible promoter enhances root growth and abiotic stress tolerance in rice without yield penalty.

    PubMed

    Chen, Yi-Shih; Lo, Shuen-Fang; Sun, Peng-Kai; Lu, Chung-An; Ho, Tuan-Hua D; Yu, Su-May

    2015-01-01

    Regulation of root architecture is essential for maintaining plant growth under adverse environment. A synthetic abscisic acid (ABA)/stress-inducible promoter was designed to control the expression of a late embryogenesis abundant protein (HVA1) in transgenic rice. The background of HVA1 is low but highly inducible by ABA, salt, dehydration and cold. HVA1 was highly accumulated in root apical meristem (RAM) and lateral root primordia (LRP) after ABA/stress treatments, leading to enhanced root system expansion. Water-use efficiency (WUE) and biomass also increased in transgenic rice, likely due to the maintenance of normal cell functions and metabolic activities conferred by HVA1 which is capable of stabilizing proteins, under osmotic stress. HVA1 promotes lateral root (LR) initiation, elongation and emergence and primary root (PR) elongation via an auxin-dependent process, particularly by intensifying asymmetrical accumulation of auxin in LRP founder cells and RAM, even under ABA/stress-suppressive conditions. We demonstrate a successful application of an inducible promoter in regulating the spatial and temporal expression of HVA1 for improving root architecture and multiple stress tolerance without yield penalty.

  14. Somatic tetraploidy in specific chick retinal ganglion cells induced by nerve growth factor

    PubMed Central

    Morillo, Sandra M.; Escoll, Pedro; de la Hera, Antonio; Frade, José M.

    2009-01-01

    A subset of neurons in the normal vertebrate nervous system contains double the normal amount of DNA in their nuclei. These neurons are all thought to derive from aberrant mitoses in neuronal precursor cells. Here we show that endogenous NGF induces DNA replication in a subpopulation of differentiating chick retinal ganglion cells that express both the neurotrophin receptor p75 and the E2F1 transcription factor, but that lack the retinoblastoma protein. Many of these neurons avoid G2/M transition and remain alive in the retina as tetraploid cells with large cell somas and extensive dendritic trees, and most of them express β2 nicotinic acetylcholine receptor subunits, a specific marker of retinal ganglion cells innervating lamina F in the stratum-griseum-et-fibrosum-superficiale of the tectal cortex. Tetraploid neurons were also observed in the adult mouse retina. Thus, a developmental program leading to somatic tetraploidy in specific retinal neurons exists in vertebrates. This program might occur in other vertebrate neurons during normal or pathological situations. PMID:20018664

  15. Characterization of immunoglobulin gene somatic hypermutation in the absence of activation-induced cytidine deaminase

    PubMed Central

    Longo, Nancy S.; Satorius, Colleen L.; Plebani, Alessandro; Durandy, Anne; Lipsky, Peter E.

    2008-01-01

    Somatic hypermutation (SHM) of Ig genes depends upon the deamination of C nucleotides in WRCY (W=A/T, R=A/G, Y=C/T) motifs by activation-induced cytidine deaminase (AICDA). Despite this, a large number of mutations occur in WA motifs that can be accounted for by the activity of polymerase eta (POL η). To determine whether there are AICDA-independent mutations and to characterize the relationship between AICDA- and POL η-mediated mutations, 1,470 H chain and 1,313 kappa and lambda chain rearrangements from three AICDA−/− patients were analyzed. The Ig mutation frequency of all VH genes from AICDA−/− patients was 40-fold less than that of normal donors whereas the mutation frequency of mutated VH sequences from AICDA−/− patients was 6.8-fold less than normal donors. AICDA−/− B cells lack mutations in WRCY/RGYW motifs as well as replacement mutations and mutational targeting in complementarity determining regions. A significantly reduced mutation frequency in WA motifs compared to normal donors and an increased percentage of transitions, which may relate to reduced uracil DNA-glycosylase (UNG) activity, suggest a role for AICDA in regulating POL η and UNG activity. Similar results were observed in VL rearrangements. The residual mutations were predominantly G:C substitutions, indicating that AICDA-independent cytidine deamination was a likely, yet inefficient, mechanism for mutating Ig genes. PMID:18606684

  16. Trichostatin A Rescues the Disrupted Imprinting Induced by Somatic Cell Nuclear Transfer in Pigs

    PubMed Central

    Huan, Yanjun; Zhu, Jiang; Huang, Bo; Mu, Yanshuang; Kong, Qingran; Liu, Zhonghua

    2015-01-01

    Imprinting disorders induced by somatic cell nuclear transfer (SCNT) usually lead to the abnormalities of cloned animals and low cloning efficiency. Histone deacetylase inhibitors have been shown to improve gene expression, genomic methylation reprogramming and the development of cloned embryos, however, the imprinting statuses in these treated embryos and during their subsequent development remain poorly studied. In this study, we investigated the dynamics of H19/Igf2 methylation and transcription in porcine cloned embryos treated with trichostatin A (TSA), and examined H19/Igf2 imprinting patterns in cloned fetuses and piglets. Our results showed that compared with the maintenance of H19/Igf2 methylation in fertilized embryos, cloned embryos displayed aberrant H19/Igf2 methylation and lower H19/Igf2 transcripts. When TSA enhanced the development of cloned embryos, the disrupted H19/Igf2 imprinting was largely rescued in these treated embryos, more similar to those detected in fertilized counterparts. Further studies displayed that TSA effectively rescued the disrupted imprinting of H19/Igf2 in cloned fetuses and piglets, prevented the occurrence of cloned fetus and piglet abnormalities, and enhanced the full-term development of cloned embryos. In conclusion, our results demonstrated that aberrant imprinting induced by SCNT led to the abnormalities of cloned fetuses and piglets and low cloning efficiency, and TSA rescued the disrupted imprinting in cloned embryos, fetuses and piglets, and prevented the occurrence of cloned fetus and piglet abnormalities, thereby improving the development of cloned embryos. This study would have important implications in improving cloning efficiency and the health of cloned animals. PMID:25962071

  17. Molecular cloning and expression of hardening-induced genes in Chlorella vulgaris C-27: the most abundant clone encodes a late embryogenesis abundant protein.

    PubMed

    Joh, T; Honjoh, K; Yoshimoto, M; Funabashi, J; Miyamoto, T; Hatano, S

    1995-01-01

    To investigate the effects of hardening on gene expression in Chlorella vulgaris Beijerink IAM C-27 (formerly Chlorella ellipsoidea Gerneck IAM C-27), a frost-hardy strain, 17 cDNA clones corresponding to hardening-induced Chlorella (hiC) genes were isolated by differential screening of a cDNA library from 6-h hardened cells. Northern blot analysis of transcripts of hiC genes showed that these genes are specifically induced by hardening and that their patterns of induction vary. Southern blots of genomic DNAs from two strains (Chlorella ellipsoidea Gerneck IAM C-102, chilling-sensitive; and C. vulgaris C-27, frost-hardy) of Chlorella indicated that ten hiC clones out of 17 hybridized only with DNA of strain C-27 and the other seven clones hybridized with DNA of both strains. However, of these seven clones, transcripts corresponding to six clones did not accumulate in strain C-102 at low temperatures. The sequence of a deduced protein encoded by the most abundant clone, hiC6, exhibited homology to sequences of Group III LEA (late embryogenesis abundant) proteins and had an amino-terminal amino acid sequence that was similar to the sequences of chloroplast transit peptides. PMID:7719632

  18. How microspores transform into haploid embryos: changes associated with embryogenesis induction and microspore-derived embryogenesis.

    PubMed

    Seguí-Simarro, José M; Nuez, Fernando

    2008-09-01

    Microspore embryogenesis is the most powerful androgenic pathway to produce haploid and doubled haploid plants. To deviate a microspore toward embryogenesis, a number of factors, different for each species, must concur at the same time and place. Once induced, the microspore undergoes numerous changes at different levels, from overall morphology to gene expression. Induction of microspore embryogenesis not only implies the expression of an embryogenic program, but also a stress-related cellular response and a repression of the gametophytic program to revert the microspore to a totipotent status. In this review, we compile the most recent advances in the understanding of the changes undergone by the induced microspore to readapt to the new developmental scenario. We devote special attention to the efforts made to uncover changes in the transcriptome of the induced microspore and microspore-derived embryo (MDE). Finally, we discuss the influence that an in vitro environment exerts over the MDE, as compared with its zygotic counterpart.

  19. Inducing pluripotency in somatic cells from the snow leopard (Panthera uncia), an endangered felid.

    PubMed

    Verma, R; Holland, M K; Temple-Smith, P; Verma, P J

    2012-01-01

    Induced pluripotency is a new approach to produce embryonic stem-like cells from somatic cells that provides a unique means to understand both pluripotency and lineage assignment. To investigate whether this technology could be applied to endangered species, where the limited availability of gametes makes production and research on embryonic stem cells difficult, we attempted generation of induced pluripotent stem (iPS) cells from snow leopard (Panthera uncia) fibroblasts by retroviral transfection with Moloney-based retroviral vectors (pMXs) encoding four factors (OCT4, SOX2, KLF4 and cMYC). This resulted in the formation of small colonies of cells, which could not be maintained beyond four passages (P4). However, addition of NANOG, to the transfection cocktail produced stable iPS cell colonies, which formed as early as D3. Colonies of cells were selected at D5 and expanded in vitro. The resulting cell line was positive for alkaline phosphatase (AP), OCT4, NANOG, and Stage-Specific embryonic Antigen-4 (SSEA-4) at P14. RT-PCR also confirmed that endogenous OCT4 and NANOG were expressed by snow leopard iPS cells from P4. All five human transgenes were transcribed at P4, but OCT4, SOX2 and NANOG transgenes were silenced as early as P14; therefore, reprogramming of the endogenous pluripotent genes had occurred. When injected into immune-deficient mice, snow leopard iPS cells formed teratomas containing tissues representative of the three germ layers. In conclusion, this was apparently the first derivation of iPS cells from the endangered snow leopard and the first report on induced pluripotency in felid species. Addition of NANOG to the reprogramming cocktail was essential for derivation of iPS lines in this felid. The iPS cells provided a unique source of pluripotent cells with utility in conservation through cryopreservation of genetics, as a source of reprogrammed donor cells for nuclear transfer or for directed differentiation to gametes in the future.

  20. Inducing pluripotency in somatic cells from the snow leopard (Panthera uncia), an endangered felid.

    PubMed

    Verma, R; Holland, M K; Temple-Smith, P; Verma, P J

    2012-01-01

    Induced pluripotency is a new approach to produce embryonic stem-like cells from somatic cells that provides a unique means to understand both pluripotency and lineage assignment. To investigate whether this technology could be applied to endangered species, where the limited availability of gametes makes production and research on embryonic stem cells difficult, we attempted generation of induced pluripotent stem (iPS) cells from snow leopard (Panthera uncia) fibroblasts by retroviral transfection with Moloney-based retroviral vectors (pMXs) encoding four factors (OCT4, SOX2, KLF4 and cMYC). This resulted in the formation of small colonies of cells, which could not be maintained beyond four passages (P4). However, addition of NANOG, to the transfection cocktail produced stable iPS cell colonies, which formed as early as D3. Colonies of cells were selected at D5 and expanded in vitro. The resulting cell line was positive for alkaline phosphatase (AP), OCT4, NANOG, and Stage-Specific embryonic Antigen-4 (SSEA-4) at P14. RT-PCR also confirmed that endogenous OCT4 and NANOG were expressed by snow leopard iPS cells from P4. All five human transgenes were transcribed at P4, but OCT4, SOX2 and NANOG transgenes were silenced as early as P14; therefore, reprogramming of the endogenous pluripotent genes had occurred. When injected into immune-deficient mice, snow leopard iPS cells formed teratomas containing tissues representative of the three germ layers. In conclusion, this was apparently the first derivation of iPS cells from the endangered snow leopard and the first report on induced pluripotency in felid species. Addition of NANOG to the reprogramming cocktail was essential for derivation of iPS lines in this felid. The iPS cells provided a unique source of pluripotent cells with utility in conservation through cryopreservation of genetics, as a source of reprogrammed donor cells for nuclear transfer or for directed differentiation to gametes in the future. PMID

  1. 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

  2. Somatic-cell mutation induced by short exposures to cigarette smoke in urate-null, oxidative stress-sensitive Drosophila.

    PubMed

    Uchiyama, Tomoyo; Koike, Ryota; Yuma, Yoko; Okamoto, Keinosuke; Arimoto-Kobayashi, Sakae; Suzuki, Toshinori; Negishi, Tomoe

    2016-01-01

    We previously reported that a urate-null strain of Drosophila is hypersensitive to cigarette smoke (CS), and we suggested that CS induces oxidative stress in Drosophila because uric acid is a potent antioxidant. Although the carcinogenic risk of CS exposure is widely recognized; documentation of in vivo genotoxic activity of environmental CS, especially gaseous-phase CS, remains inconclusive. To date, somatic-cell mutations in Drosophila resulting from exposure to CS have not been detected via the somatic mutation and recombination test (wing spot test) with wild-type flies, a widely used Drosophila assay for the detection of somatic-cell mutation; moreover, genotoxicity has not been documented via a DNA repair test that involves DNA repair-deficient Drosophila. In this study, we used a new Drosophila strain (y v ma-l; mwh) to examine the mutagenicity induced by gaseous-phase CS; these flies are urate-null due to a mutation in ma-l, and they are heterozygous for multiple wing hair (mwh), a mutation that functions as a marker for somatic-cell mutation. In an assay with this newly developed strain, a superoxide anion-producing weed-killer, paraquat, exhibited significant mutagenicity; in contrast, paraquat was hardly mutagenic with a wild-type strain. Drosophila larvae were exposed to CS for 2, 4 or 6h, and then kept at 25°C on instant medium until adulthood. After eclosion, mutant spots, which consisted of mutant hairs on wings, were scored. The number of mutant spots increased significantly in an exposure time-dependent manner in the urate-null females (ma-l (-/-)), but not in the urate-positive females (ma-l (+/-)). In this study, we showed that short-term exposure to CS was mutagenic in this in vivo system. In addition, we obtained suggestive data regarding reactive oxygen species production in larva after CS exposure using the fluorescence probe H2DCFDA. These results suggest that oxidative damage, which might be countered by uric acid, was partly responsible

  3. Induced malignant genome reprogramming in somatic cells by testis-specific factors.

    PubMed

    Wang, Jin; Emadali, Anouk; Le Bescont, Aurore; Callanan, Mary; Rousseaux, Sophie; Khochbin, Saadi

    2011-01-01

    Germline cell differentiation is controlled by a specific set of genes whose expression is tightly locked into the repressed state in somatic cells. Large-scale epigenome alterations, now evidenced in nearly all cancers, lead to aberrant activation of these normally silenced genes, as attested by the many reports describing the expression of testis-specific factors, known as cancer-testis genes, in various cancer cells. Here, based on the literature, we argue that off-context activity of some of the testis-specific epigenome regulators can reprogram the somatic cell epigenome toward a malignant state by favoring self-renewal and sustaining cell proliferation under stressful conditions, thereby constituting a major oncogenic mechanism.

  4. Xist repression shows time-dependent effects on the reprogramming of female somatic cells to induced pluripotent stem cells.

    PubMed

    Chen, Qi; Gao, Shuai; He, Wenteng; Kou, Xiaochen; Zhao, Yanhong; Wang, Hong; Gao, Shaorong

    2014-10-01

    Although the reactivation of silenced X chromosomes has been observed as part of the process of reprogramming female somatic cells into induced pluripotent stem cells (iPSCs), it remains unknown whether repression of the X-inactive specific transcript (Xist) can greatly enhance female iPSC induction similar to that observed in somatic cell nuclear transfer studies. In this study, we discovered that the repression of Xist plays opposite roles in the early and late phases of female iPSCs induction. Our results demonstrate that the downregulation of Xist by an isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible short hairpin RNA (shRNA) system can greatly impair the mesenchymal-to-epithelial transition (MET) in the early phase of iPSC induction but can significantly promote the transition of pre-iPSCs to iPSCs in the late phase. Furthermore, we demonstrate that although the knockdown of Xist did not affect the H3K27me3 modification on the X chromosome, macroH2A was released from the inactivated X chromosome (Xi). This enables the X chromosome silencing to be a reversible event. Moreover, we demonstrate that the supplementation of vitamin C (Vc) can augment and stabilize the reversible X chromosome by preventing the relocalization of macroH2A to the Xi. Therefore, our study reveals an opposite role of Xist repression in the early and late stages of reprogramming female somatic cells to pluripotency and demonstrates that the release of macroH2A by Xist repression enables the transition from pre-iPSCs to iPSCs.

  5. Late embryogenesis abundant proteins

    PubMed Central

    Olvera-Carrillo, Yadira; Reyes, José Luis

    2011-01-01

    Late Embryogenesis Abundant (LEA) proteins accumulate at the onset of seed desiccation and in response to water deficit in vegetative plant tissues. The typical LEA proteins are highly hydrophilic and intrinsically unstructured. They have been classified in different families, each one showing distinctive conserved motifs. In this manuscript we present and discuss some of the recent findings regarding their role in plant adaptation to water deficit, as well as those concerning to their possible function, and how it can be related to their intrinsic structural flexibility. PMID:21447997

  6. Distribution of poly(A)-containing RNA during normal pollen development and during induced pollen embryogenesis in Hyoscyamus niger

    PubMed Central

    1981-01-01

    The distribution of poly(A)-containing RNA [poly(A)+RNA] in pollen grains of Hyoscyamus niger during normal gametophytic development and embryogenic development induced by culture of anther segments was followed by in situ hybridization with [3H]-polyuridylic acid as a probe. No binding of the isotope occurred in pollen grains during the uninucleate phase of their development. Although [3H]polyuridylic acid binding sites were present in the generative and vegetative cells of maturing pollen grains, they almost completely disappeared from mature grains ready to germinate. During pollen germination, poly(A)+RNA formation was transient and was due to the activity of the generative nucleus, whereas the vegetative nucleus and the sperm cells failed to interact with the applied probe. In cultured anther segments, moderate amounts of poly(A)+RNA were detected in the uninucleate, nonvacuolate, embryogenically determined pollen grains. Poly(A)+RNA accumulation in these grains was sensitive to actinomycin D, suggesting that it represents newly transcribed mRNA. After the first haploid mitosis in the embryogenically determined pollen grains, only those grains in which the generative nucleus alone or along with the vegetative nucleus accumulated poly(A)+RNA in the surrounding cytoplasm were found to divide in the embryogenic pathway. Overall, the results suggest that, in contrast to normal gametophytic development, embryogenic development in the uninucleate pollen grains of cultured anther segments of H. niger is due to the transcriptional activation of an informational type of RNA. Subsequent divisions in the potentially embryogenic binucleate pollen grains appeared to be mediated by the continued synthesis of mRNA either in the generative nucleus or in both the generative and vegetative nuclei. PMID:6166618

  7. Oncogenic transformation of Drosophila somatic cells induces a functional piRNA pathway.

    PubMed

    Fagegaltier, Delphine; Falciatori, Ilaria; Czech, Benjamin; Castel, Stephane; Perrimon, Norbert; Simcox, Amanda; Hannon, Gregory J

    2016-07-15

    Germline genes often become re-expressed in soma-derived human cancers as "cancer/testis antigens" (CTAs), and piRNA (PIWI-interacting RNA) pathway proteins are found among CTAs. However, whether and how the piRNA pathway contributes to oncogenesis in human neoplasms remain poorly understood. We found that oncogenic Ras combined with loss of the Hippo tumor suppressor pathway reactivates a primary piRNA pathway in Drosophila somatic cells coincident with oncogenic transformation. In these cells, Piwi becomes loaded with piRNAs derived from annotated generative loci, which are normally restricted to either the germline or the somatic follicle cells. Negating the pathway leads to increases in the expression of a wide variety of transposons and also altered expression of some protein-coding genes. This correlates with a reduction in the proliferation of the transformed cells in culture, suggesting that, at least in this context, the piRNA pathway may play a functional role in cancer. PMID:27474441

  8. Diethylnitrosamine-induced expression of germline-specific genes and pluripotency factors, including vasa and oct4, in medaka somatic cells.

    PubMed

    Shen, Jialing; Yokota, Shinpei; Yokoi, Hayato; Suzuki, Tohru

    2016-09-16

    Various methods have been developed to reprogram mammalian somatic cells into pluripotent cells as well as to directly reprogram somatic cells into other cell lineages. We are interested in applying these methods to fish, and here, we examined whether mRNA expression of germline-specific genes (vasa, nanos2, -3) and pluripotency factors (oct4, sox2, c-myc, nanog) is inducible in somatic cells of Japanese medaka (Oryzias latipes). We found that the expression of vasa is induced in the gut and regenerating fin by exposure to a carcinogen, diethylnitrosamine (DEN). Induction of vasa in the gut started on the 5th day of treatment with >50 ppm DEN. In addition, nanos2, -3, oct4, sox2, klf4, c-myc, and nanog were also expressed simultaneously in some vasa-positive gut and regenerating fin samples. Vasa-positive cells were detected by immunohistochemistry (IHC) in the muscle surrounding the gut and in the wound epidermis, blastema, and fibroblast-like cells in regenerating fin. In vasa:GFP transgenic medaka, green fluorescent protein (GFP) fluorescence appeared in the wound epidermis and fibroblast-like cells in the regenerating fin following DEN exposure, in agreement with the IHC data. Our data show that mRNA expression of genes relevant to germ cell specification and pluripotency can be induced in fish somatic cells by exposure to DEN, suggesting the possibility of efficient and rapid cell reprogramming of fish somatic cells. PMID:27514449

  9. [Mechanisms of adrenal embryogenesis].

    PubMed

    Barinov, E F; Sulaeva, O N

    2001-01-01

    The aim of this vie is to discuss the general principles of prenatal development of adrenal gland. On the basis of spatial-temporal heterogenity of structural particularites of fetal adrenal cortex, spectrum steroidogenic enzymes and secreting hormones expression in adrenocorticocytes, regulation of proliferation and differentiation processes mechanisms, authors discuss adrenal morphogenesis in three periods of gestation. It was noted the close relationship between placenta development and hypothalamo-pituitary-adrenocortical system formation with specification in each gestation period. Adrenal embryogenesis accompanied by remodeling of structural, functional and biochemical properties of parenchimal-stromal elements of fetal organ. Definitive zonation formation determined by morphogens: ACTH, renal and intraadrenal angiotensin II, estrogens, prostaglandines and other. The action of these factors realization is due to immediately and thought growth factor system (IGF-I, IGF-II, EGF, bFGF), working as paracrine amplificators of morphogenetic signals and activators of transcriptional factors--c-fos and c-jun.

  10. 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

  11. 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

  12. 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

    2016-02-23

    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.

  13. New theory of uterovaginal embryogenesis

    PubMed Central

    Makiyan, Zograb

    2016-01-01

    ABSTRACT Background: The explanation of uterine and vaginal embryogenesis in humans still poses many controversies, because it is difficult to assess early stages of an embryo. The literature review revealed many disagreements in Mullerian theory, inciting some authors to propose new embryological hypotheses. In the original Mullerian theory: the paramesonephral ducts form the Fallopian tubes, uterus and vagina; the mesonephral ducts regress in female embryos. Aims: The aim of this article is to investigate the development of Mullerian ducts in humans, using comparative analysis of fundamental embryological theory and various utero-vaginal anomalies. Material and methods: Between 1998 and 2015, 434 patients with various uterovaginal malformations had been operated on at the Scientific Centre of Obstetrics Gynaecology and Perynatology in Moscow. The anatomies of the uterovaginal malformations in these patients were diagnosed with ultrasound and MRI and then verified during surgical correction by laparoscopy. Results: A systematic comparison of uterovaginal malformations to those in the literature has allowed us to formulate a new theory of embryonic morphogenesis. The new theory is significantly different: ovary, ovarian ligamentum proprium, and ligamentum teres uteri derive from gonadal ridges; Fallopian tubes and vagina completely develop from mesonephral ducts. The uterus develops in the area of intersection between the mesonephral ducts with gonadal ridges by the fusion of the two. Conclusions: The new theory may to induce future embryological studies. The hypothetic possibility that the ovary and endometrium derive from the gonadal ridges could be the key to understanding the enigmatic aetiologies of extragenital and ovarian endometriosis. PMID:26900909

  14. Assessment of imidacloprid-induced mutagenic effects in somatic cells of Swiss albino male mice.

    PubMed

    Bagri, Preeti; Kumar, Vinod; Sikka, Anil K

    2016-10-01

    Pesticides are being used for plant protection to increase food protection and to reduce insect-borne diseases worldwide. Exposure to the pesticides may cause genotoxic effects on both the target and nontarget organisms, including man. Therefore, the mutagenicity evaluation of such pesticides has become a priority area of research. Imidacloprid (IMI), a neonicotinoid insecticide, is widely used in agriculture either alone or in combination with other insecticides. A combined approach employing micronucleus test (MNT) and chromosomal aberrations assay (CA) was utilized to assess the mutagenicity of imidacloprid in bone marrow of Swiss albino male mice. IMI suspension was prepared in 3% gum acacia and administered at doses of 5.5, 11 and 22 mg/kg body weight for 7, 14 and 28 days to mice. IMI treatment resulted in a dose and time-dependant increase in the frequencies of micronuclei per cell and chromosomal aberrations in bone marrow cells. A statistically significant increase in chromosomal aberrations and micronuclei/cell was found only after daily treatment of IMI at highest selected dose (22 mg/kg body weight) for longest selected time period (28 days) compared to the control group. Thus, daily exposure of imidacloprid at a dose level of 22 mg/kg body weight for 28 days caused mutagenic effects on the somatic cells of Swiss albino male mice.

  15. Assessment of imidacloprid-induced mutagenic effects in somatic cells of Swiss albino male mice.

    PubMed

    Bagri, Preeti; Kumar, Vinod; Sikka, Anil K

    2016-10-01

    Pesticides are being used for plant protection to increase food protection and to reduce insect-borne diseases worldwide. Exposure to the pesticides may cause genotoxic effects on both the target and nontarget organisms, including man. Therefore, the mutagenicity evaluation of such pesticides has become a priority area of research. Imidacloprid (IMI), a neonicotinoid insecticide, is widely used in agriculture either alone or in combination with other insecticides. A combined approach employing micronucleus test (MNT) and chromosomal aberrations assay (CA) was utilized to assess the mutagenicity of imidacloprid in bone marrow of Swiss albino male mice. IMI suspension was prepared in 3% gum acacia and administered at doses of 5.5, 11 and 22 mg/kg body weight for 7, 14 and 28 days to mice. IMI treatment resulted in a dose and time-dependant increase in the frequencies of micronuclei per cell and chromosomal aberrations in bone marrow cells. A statistically significant increase in chromosomal aberrations and micronuclei/cell was found only after daily treatment of IMI at highest selected dose (22 mg/kg body weight) for longest selected time period (28 days) compared to the control group. Thus, daily exposure of imidacloprid at a dose level of 22 mg/kg body weight for 28 days caused mutagenic effects on the somatic cells of Swiss albino male mice. PMID:26823062

  16. Modulatory effects of Tabebuia impetiginosa (Lamiales, Bignoniaceae) on doxorubicin-induced somatic mutation and recombination in Drosophila melanogaster

    PubMed Central

    2009-01-01

    The wing Somatic Mutation and Recombination Test (SMART) in D. melanogaster was used to study genotoxicity of the medicinal plant Tabebuia impetiginosa. Lapachol (naphthoquinone) and β-lapachone (quinone) are the two main chemical constituents of T. impetiginosa. These compounds have several biological properties. They induce apoptosis by generating oxygen-reactive species, thereby inhibiting topoisomerases (I and II) or inducing other enzymes dependent on NAD(P)H:quinone oxidoreductase 1, thus affecting cell cycle checkpoints. The SMART was used in the standard (ST) version, which has normal levels of cytochrome P450 (CYP) enzymes, to check the direct action of this compound, and in the high bioactivation (HB) version, which has a high constitutive level of CYP enzymes, to check for indirect action in three different T. impetiginosa concentrations (10%, 20% or 40% w/w). It was observed that T. impetiginosa alone did not modify the spontaneous frequencies of mutant spots in either cross. The negative results observed prompted us to study this phytotherapeuticum in association with the reference mutagen doxorubicin (DXR). In co-treated series, T. impetiginosa was toxic in both crosses at higher concentration, whereas in the HB cross, it induced a considerable potentiating effect (from ~24.0 to ~95.0%) on DXR genotoxity. Therefore, further research is needed to determine the possible risks associated with the exposure of living organisms to this complex mixture. PMID:21637695

  17. Siberian Sturgeon Oocyte Extract Induces Epigenetic Modifications of Porcine Somatic Cells and Improves Developmental Competence of SCNT Embryos

    PubMed Central

    Kim, So-Young; Kim, Tae-Suk; Park, Sang-Hoon; Lee, Mi-Ran; Eun, Hye-Ju; Baek, Sang-Ki; Ko, Yeoung-Gyu; Kim, Sung-Woo; Seong, Hwan-Hoo; Campbell, Keith H.S.; Lee, Joon-Hee

    2014-01-01

    Somatic cell nuclear transfer (SCNT) has generally demonstrated that a differentiated cell can convert into a undifferentiated or pluripotent state. In the SCNT experiment, nuclear reprogramming is induced by exposure of introduced donor nuclei to the recipient cytoplasm of matured oocytes. However, because the efficiency of SCNT still remains low, a combination of SCNT technique with the ex-ovo method may improve the normal development of SCNT embryos. Here we hypothesized that treatment of somatic cells with extracts prepared from the germinal vesicle (GV) stage Siberian sturgeon oocytes prior to their use as nuclear donor for SCNT would improve in vitro development. A reversible permeability protocol with 4 μg/mL of digitonin for 2 min at 4°C in order to deliver Siberian sturgeon oocyte extract (SOE) to porcine fetal fibroblasts (PFFs) was carried out. As results, the intensity of H3K9ac staining in PFFs following treatment of SOE for 7 h at 18°C was significantly increased but the intensity of H3K9me3 staining in PFFs was significantly decreased as compared with the control (p<0.05). Additionally, the level of histone acetylation in SCNT embryos at the zygote stage was significantly increased when reconstructed using SOE-treated cells (p<0.05), similar to that of IVF embryos at the zygote stage. The number of apoptotic cells was significantly decreased and pluripotency markers (Nanog, Oct4 and Sox2) were highly expressed in the blastocyst stage of SCNT embryos reconstructed using SOE-treated cells as nuclear donor (p<0.05). And there was observed a better development to the blastocyst stage in the SOE-treated group (p<0.05). Our results suggested that pre-treatment of cells with SOE could improve epigenetic reprogramming and the quality of porcine SCNT embryos. PMID:25049951

  18. Modulating effect of simvastatin on the DNA damage induced by doxorubicin in somatic cells of Drosophila melanogaster.

    PubMed

    Orsolin, P C; Silva-Oliveira, R G; Nepomuceno, J C

    2016-04-01

    Simvastatin is an antilipemic drug that promotes inhibition of HMG-CoA reductase. Simvastatin can also inhibit the formation of other products, such as isoprenoids, conferring additional benefits to this drug, which include antiproliferative, anti-invasive and pro-apoptotic effects. This study was carried out with the aim of evaluating the mutagenic/recombinogenic effect of simvastatin as well as the possible modulatory effects of this statin on the DNA damage induced by doxorubicin (DXR). This analysis was performed using the somatic mutation and recombination test (SMART) in Drosophila melanogaster. To study these effects, larvae descendants of both crosses (ST and HB) were chronically treated with five concentrations of simvastatin, separately and in association with DXR. The results revealed no mutagenic/recombinogenic effect of simvastatin for any of the concentrations tested. A modulating effect of simvastatin was also observed on DNA damage induced by DXR. The reduction of total mutant frequency was observed for spots from descendants of both crosses, but the inhibition was more effective in descendants from the standard cross (ST). It is believed that this modulating effect is mainly associated with the antioxidant activity of this class of drugs, although this parameter has not been directly assessed in this study. PMID:26829615

  19. Neuregulin-1 Administration Protocols Sufficient for Stimulating Cardiac Regeneration in Young Mice Do Not Induce Somatic, Organ, or Neoplastic Growth

    PubMed Central

    Ganapathy, Balakrishnan; Nandhagopal, Nikitha; Polizzotti, Brian D.; Bennett, David; Asan, Alparslan; Wu, Yijen; Kühn, Bernhard

    2016-01-01

    Background We previously developed and validated a strategy for stimulating heart regeneration by administration of recombinant neuregulin (rNRG1), a growth factor, in mice. rNRG1 stimulated proliferation of heart muscle cells, cardiomyocytes, and was most effective when administration began during the neonatal period. Our results suggested the use of rNRG1 to treat pediatric patients with heart failure. However, administration in this age group may stimulate growth outside of the heart. Methods NRG1 and ErbB receptor expression was determined by RT-PCR. rNRG1 concentrations in serum were quantified by ELISA. Mice that received protocols of recombinant neuregulin1-β1 administration (rNRG1, 100 ng/g body weight, daily subcutaneous injection for the first month of life), previously shown to induce cardiac regeneration, were examined at pre-determined intervals. Somatic growth was quantified by weighing. Organ growth was quantified by MRI and by weighing. Neoplastic growth was examined by MRI, visual inspection, and histopathological analyses. Phospho-ERK1/2 and S6 kinase were analyzed with Western blot and ELISA, respectively. Results Lung, spleen, liver, kidney, brain, and breast gland exhibited variable expression of the NRG1 receptors ErbB2, ErbB3, ErbB4, and NRG1. Body weight and tibia length were not altered in mice receiving rNRG1. MRI showed that administration of rNRG1 did not alter the volume of the lungs, liver, kidneys, brain, or spinal cord. Administration of rNRG1 did not alter the weight of the lungs, spleen, liver, kidneys, or brain. MRI, visual inspection, and histopathological analyses showed no neoplastic growth. Follow-up for 6 months showed no alteration of somatic or organ growth. rNRG1 treatment increased the levels of phospho-ERK1/2, but not phospho-S6 kinase. Conclusions Administration protocols of rNRG1 for stimulating cardiac regeneration in mice during the first month of life did not induce unwanted growth effects. Further studies may be

  20. PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells.

    PubMed

    Chandrakanthan, Vashe; Yeola, Avani; Kwan, Jair C; Oliver, Rema A; Qiao, Qiao; Kang, Young Chan; Zarzour, Peter; Beck, Dominik; Boelen, Lies; Unnikrishnan, Ashwin; Villanueva, Jeanette E; Nunez, Andrea C; Knezevic, Kathy; Palu, Cintia; Nasrallah, Rabab; Carnell, Michael; Macmillan, Alex; Whan, Renee; Yu, Yan; Hardy, Philip; Grey, Shane T; Gladbach, Amadeus; Delerue, Fabien; Ittner, Lars; Mobbs, Ralph; Walkley, Carl R; Purton, Louise E; Ward, Robyn L; Wong, Jason W H; Hesson, Luke B; Walsh, William; Pimanda, John E

    2016-04-19

    Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor-AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.

  1. Generation of porcine fetal fibroblasts expressing the tetracycline-inducible Cas9 gene by somatic cell nuclear transfer.

    PubMed

    Liu, Guoqian; Liu, Kai; Wei, Hengxi; Li, Li; Zhang, Shouquan

    2016-09-01

    Cas9 endonuclease, from so-called clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems of Streptococcus pyogenes, type II functions as an RNA-guided endonuclease and edits the genomes of prokaryotic and eukaryotic organisms, including deletion and insertion by DNA double‑stranded break repair mechanisms. In previous studies, it was observed that Cas9, with a genome‑scale lentiviral single‑guide RNA library, could be applied to a loss‑of‑function genetic screen, although the loss‑of‑function genes have yet to be verified in vitro and this approach has not been used in porcine cells. Based on these observations, lentiviral Cas9 was used to infect porcine primary fibroblasts to achieve cell colonies carrying Cas9 endonuclease. Subsequently, porcine fetal fibroblasts expressing the tetracycline‑inducible Cas9 gene were generated by somatic cell nuclear transfer, and three 30 day transgenic porcine fetal fibroblasts (PFFs) were obtained. Polymerase chain reaction (PCR), reverse transcription‑PCR and western blot analysis indicated that the PFFs were Cas9‑positive. In addition, one of the three integrations was located near to known functional genes in the PFF1 cell line, whereas neither of the integrations was located in the PFF1 or PFF2 cell lines. It was hypothesized that these transgenic PFFs may be useful for conditional genomic editing in pigs, and for generating ideal modified porcine models. PMID:27430306

  2. Generation of porcine fetal fibroblasts expressing the tetracycline-inducible Cas9 gene by somatic cell nuclear transfer

    PubMed Central

    Liu, Guoqian; Liu, Kai; Wei, Hengxi; Li, Li; Zhang, Shouquan

    2016-01-01

    Cas9 endonuclease, from so-called clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems of Streptococcus pyogenes, type II functions as an RNA-guided endonuclease and edits the genomes of prokaryotic and eukaryotic organisms, including deletion and insertion by DNA double-stranded break repair mechanisms. In previous studies, it was observed that Cas9, with a genome-scale lentiviral single-guide RNA library, could be applied to a loss-of-function genetic screen, although the loss-of-function genes have yet to be verified in vitro and this approach has not been used in porcine cells. Based on these observations, lentiviral Cas9 was used to infect porcine primary fibroblasts to achieve cell colonies carrying Cas9 endonuclease. Subsequently, porcine fetal fibroblasts expressing the tetracycline-inducible Cas9 gene were generated by somatic cell nuclear transfer, and three 30 day transgenic porcine fetal fibroblasts (PFFs) were obtained. Polymerase chain reaction (PCR), reverse transcription-PCR and western blot analysis indicated that the PFFs were Cas9-positive. In addition, one of the three integrations was located near to known functional genes in the PFF1 cell line, whereas neither of the integrations was located in the PFF1 or PFF2 cell lines. It was hypothesized that these transgenic PFFs may be useful for conditional genomic editing in pigs, and for generating ideal modified porcine models. PMID:27430306

  3. Can Body Condition and Somatic Indices be Used to Evaluate Metal-Induced Stress in Wild Small Mammals?

    PubMed Central

    Tête, Nicolas; Fritsch, Clémentine; Afonso, Eve; Coeurdassier, Michaël; Lambert, Jean-Claude; Giraudoux, Patrick; Scheifler, Renaud

    2013-01-01

    Wildlife is exposed to natural (e.g., food availability and quality, parasitism) and anthropogenic stressors (e.g., habitat fragmentation, toxicants). Individual variables (e.g., age, gender) affect behaviour and physiology of animals. Together, these parameters can create both great inter-individual variations in health indicators and interpretation difficulties. We investigated the relevance of body condition and somatic indices (liver, kidneys) as indicators of health status in wood mice (Apodemus sylvaticus, n = 560) captured along a metal pollution gradient in four landscape types (30 sampling squares 500-m sided). The indices were calculated using a recently proposed standard major axis regression instead of an ordinary least square regression. After considering age and gender for the body condition index, no landscape type influence was detected in the indices. However, important index variability was observed between sampling squares; this effect was included as a random effect in linear models. After integrating all individual and environmental variables that may affect the indices, cadmium (Cd) concentrations in both the liver and kidneys were negatively related to body condition and liver indices only for individuals from highly contaminated sites. Lead in the liver was negatively related to the liver index, and Cd in kidneys was positively linked to the kidney index, potentially suggesting metal-induced stress. However, interpretation of these indices as a wildlife ecotoxicology tool should be performed with caution due to the sensitivity of potentially confounding variables (e.g., individual factors and environmental parameters). PMID:23824591

  4. Fishery-Induced Selection for Slow Somatic Growth in European Eel

    PubMed Central

    Bevacqua, Daniele; Capoccioni, Fabrizio; Melià, Paco; Vincenzi, Simone; Pujolar, José M.; De Leo, Giulio A.; Ciccotti, Eleonora

    2012-01-01

    Both theoretical and experimental studies have shown that fishing mortality can induce adaptive responses in body growth rates of fishes in the opposite direction of natural selection. We compared body growth rates in European eel (Anguilla anguilla) from three Mediterranean stocks subject to different fishing pressure. Results are consistent with the hypotheses that i) fast-growing individuals are more likely to survive until sexual maturity than slow-growing ones under natural conditions (no fishing) and ii) fishing can select for slow-growing individuals by removing fast-growing ones. Although the possibility of human-induced evolution seems remote for a panmictic species like such as the European eel, further research is desirable to assess the implications of the intensive exploitation on this critically endangered fish. PMID:22666373

  5. Modulating effect of synthetic statins against damage induced by doxorubicin in somatic cells of Drosophila melanogaster.

    PubMed

    Orsolin, P C; Silva-Oliveira, R G; Nepomuceno, J C

    2015-07-01

    The competitive inhibitors of HMG-CoA reductase, popularly known as statins, exert pleiotropic effects, which result from the ability of statins to inhibit the synthesis of isoprenoids, which are fundamental for the functioning of proteins responsible for intracellular signaling. Some recent studies suggest an important role associated with the use of antineoplastic atorvastatin and rosuvastatin, the statins most widely used today. In this study, the Drosophila wing spot test was used to evaluate possible protective effects of atorvastatin and rosuvastatin against damage induced by DXR. Larvae were chronically treated with negative control (ethanol 5%), positive control (DXR 0.125 mg/mL) and five different concentrations of atorvastatin and rosuvastatin. The results demonstrated absence of a mutagenic effect for the two statins tested. The analysis of the descendants co-treated with DXR and atorvastatin/rosuvastatin revealed a modulatory effect of these statins on damage induced by DXR. This effect was verified in all concentrations tested in the descendants of the ST and HB crosses treated with rosuvastatin, and only in descendants of the HB cross treated with atorvastatin. Induction of apoptosis and antioxidant activity appear to be the main mechanisms involved in reducing the frequency of mutant spots and consequent modulation of the damage induced by DXR. PMID:25846503

  6. [Comparative study of effect of infrared, submillimeter, and millimeter electromagnetic radiation on wing somatic mutations in Drosophila melanogaster induced by gamma-irradiation].

    PubMed

    Fedorov, V I; Pogodin, A S; Dubatolova, T D; Varlamov, A V; Leont'ev, K V; Khamoian, A G

    2001-01-01

    It was shown that the number of spontaneous and gamma-radiation-induced somatic mutations in wing cells of fruit flies (third instar larvae) exposed to laser irradiation of submillimeter range (lambda = 81.5 microns) was significantly lower than in control. Laser irradiation did not affect the number of recombinations. Exposure to laser radiation in the infrared range and electromagnetic waves of the millimeter range (lambda = 3.8 mm) enhanced the effect of gamma-irradiation.

  7. Double-strand break-induced recombination between ectopic homologous sequences in somatic plant cells.

    PubMed Central

    Puchta, H

    1999-01-01

    Homologous recombination between ectopic sites is rare in higher eukaryotes. To test whether double-strand breaks (DSBs) can induce ectopic recombination, transgenic tobacco plants harboring two unlinked, nonfunctional homologous parts of a kanamycin resistance gene were produced. To induce homologous recombination between the recipient locus (containing an I-SceI site within homologous sequences) and the donor locus, the rare cutting restriction enzyme I-SceI was transiently expressed via Agrobacterium in these plants. Whereas without I-SceI expression no recombination events were detectable, four independent recombinants could be isolated after transient I-SceI expression, corresponding to approximately one event in 10(5) transformations. After regeneration, the F1 generation of all recombinants showed Mendelian segregation of kanamycin resistance. Molecular analysis of the recombinants revealed that the resistance gene was indeed restored via homologous recombination. Three different kinds of reaction products could be identified. In one recombinant a classical gene conversion without exchange of flanking markers occurred. In the three other cases homologous sequences were transferred only to one end of the break. Whereas in three cases the ectopic donor sequence remained unchanged, in one case rearrangements were found in recipient and donor loci. Thus, ectopic homologous recombination, which seems to be a minor repair pathway for DSBs in plants, is described best by recombination models that postulate independent roles for the break ends during the repair process. PMID:10388832

  8. Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

    PubMed Central

    Choi, In Young; Lim, HoTae; Lee, Gabsang

    2014-01-01

    A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases. Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner. PMID:24798302

  9. 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

  10. 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

  11. 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.

  12. Essential elements for translation: the germline factor Vasa functions broadly in somatic cells

    PubMed Central

    Yajima, Mamiko; Wessel, Gary M.

    2015-01-01

    ABSTRACT Vasa is a conserved RNA-helicase found in the germ lines of all metazoans tested. Whereas Vasa presence is often indicated as a metric for germline determination in animals, it is also expressed in stem cells of diverse origin. Recent research suggests, however, that Vasa has a much broader function, including a significant role in cell cycle regulation. Results herein indicate that Vasa is utilized widely, and often induced transiently, during development in diverse somatic cells and adult precursor tissues. We identified that Vasa in the sea urchin is essential for: (1) general mRNA translation during embryogenesis, (2) developmental re-programming upon manipulations to the embryo and (3) larval wound healing. We also learned that Vasa interacted with mRNAs in the perinuclear area and at the spindle in an Importin-dependent manner during cell cycle progression. These results suggest that, when present, Vasa functions are essential to contributing to developmental regulation. PMID:25977366

  13. Asymmetries during molluscan embryogenesis.

    PubMed

    van den Biggelaar, J A

    1991-01-01

    In some molluscan species the unfertilized egg is symmetrical around its centre. The maturation divisions provide the egg with an axial symmetry with an animal-vegetal asymmetry. During the first two cleavages the egg loses its axial symmetry by the formation of unequal quadrants. The size differences may be very pronounced in species where the first two cleavages are accompanied by the formation of a polar lobe or where the first two cleavages are very unequal. There are some molluscan species in which at first glance the four quadrants appear equal. Exact measurements of the relative volumes have shown that the spiral character of the cleavages gives rise to minor differences between the quadrants. During further division this difference is limited to the vegetal macromeres; other corresponding blastomeres in the four quadrants are mutually equal. Therefore the absolute difference between the macromeres increases after each division. The size difference between the macromeres predisposes the biggest macromere to attain a central position and to become induced to develop the stem cell of the mesoderm. The bilateral symmetry is later lost by the counterclockwise rotation through 180 degrees of the visceral mass in relation to the head and foot. PMID:1802639

  14. Reprogramming of somatic cells induced by fusion of embryonic stem cells using hemagglutinating virus of Japan envelope (HVJ-E)

    SciTech Connect

    Yue, Xiao-shan; Fujishiro, Masako; Toyoda, Masashi; Akaike, Toshihiro; Ito, Yoshihiro

    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.

  15. Five classic articles in somatic cell reprogramming.

    PubMed

    Park, In-Hyun

    2010-09-01

    Research on somatic cell reprogramming has progressed significantly over the past few decades, from nuclear transfer into frogs' eggs in 1952 to the derivation of human-induced pluripotent stem (iPS) cells in the present day. In this article, I review five landmark papers that have laid the foundation for current efforts to apply somatic cell reprogramming in the clinic. PMID:20885901

  16. The immunodominant antigen of an ultraviolet-induced regressor tumor is generated by a somatic point mutation in the DEAD box helicase p68.

    PubMed

    Dubey, P; Hendrickson, R C; Meredith, S C; Siegel, C T; Shabanowitz, J; Skipper, J C; Engelhard, V H; Hunt, D F; Schreiber, H

    1997-02-17

    The genetic origins of CD8+ T cell-recognized unique antigens to which mice respond when immunized with syngeneic tumor cells are unknown. The ultraviolet light-induced murine tumor 8101 expresses an H-2Kb-restricted immunodominant antigen, A, that induces cytolytic CD8+ T cells in vivo A+ 8101 cells are rejected by naive mice while A- 8101 tumor cells grow. To identify the antigen H-2Kb molecules were immunoprecipitated from A+ 8101 cells and peptides were eluted by acid. The sensitizing peptide was isolated by sequential reverse-phase HPLC and sequenced using microcapillary HPLC-triple quadruple mass spectrometry. The peptide, SNFVFAGI, matched the sequence of the DEAD box protein p68 RNA helicase except for a single amino acid substitution, caused by a single nucleotide change. This mutation was somatic since fibroblasts from the mouse of tumor origin expressed the wild-type sequence. The amino acid substitution created an anchor for binding of the mutant peptide to H-2Kb. Our results are consistent with mutant p68 being responsible for rejection of the tumor. Several functions of p68, which include nucleolar assembly and inhibition of DNA unwinding, may be mediated through its IQ domain, which was altered by the mutation. This is the first description of a somatic tumor-specific mutation in the coding region of a nucleic acid helicase.

  17. Short communication: Associations between teat dimensions and milking-induced changes in teat dimensions and quarter milk somatic cell counts in dairy cows.

    PubMed

    Zwertvaegher, I; De Vliegher, S; Verbist, B; Van Nuffel, A; Baert, J; Van Weyenberg, S

    2013-02-01

    Although many studies have examined the relation between a wide range of factors and quarter milk somatic cell count (qSCC), including physical characteristics of the teat and changes in teat tissue due to milking, the effect of short-term, milking-induced changes in teat dimensions on somatic cell count has not yet been investigated. To identify teat dimensions and milking-induced changes in teat dimensions associated with qSCC, we conducted a longitudinal study (n(herds)=6, n(cows)=72, n(measurements)=12). Parity, stage of lactation, teat barrel diameter, and changes in teat barrel diameter during milking were identified as factors associated with qSCC. Teats with wider barrels had higher qSCC. Negative changes in the diameter of the teat barrel during milking (i.e., thinner teats postmilking compared with premilking) were associated with lower qSCC, whereas positive changes (i.e., thicker teats postmilking compared with premilking) were associated with higher qSCC. Selection toward more optimal teat characteristics may therefore result in improved milk quality and udder health. However, a threshold might exist for the maximum reduction in teat barrel diameter below which udder health is negatively influenced. If so, changes in teat barrel diameter might serve as an indicator for suboptimal milking and incorrect choice of teatcup liner or milking machine settings and thus help improve management of the herd. PMID:23219124

  18. The Immunodominant Antigen of an Ultraviolet-induced Regressor Tumor Is Generated by a Somatic Point Mutation in the DEAD Box Helicase p68

    PubMed Central

    Dubey, Purnima; Hendrickson, Ronald C.; Meredith, Stephen C.; Siegel, Christopher T.; Shabanowitz, Jeffrey; Skipper, Jonathan C.A.; Engelhard, Victor H.; Hunt, Donald F.; Schreiber, Hans

    1997-01-01

    The genetic origins of CD8+ T cell–recognized unique antigens to which mice respond when immunized with syngeneic tumor cells are unknown. The ultraviolet light-induced murine tumor 8101 expresses an H-2Kb-restricted immunodominant antigen, A, that induces cytolytic CD8+ T cells in vivo A+ 8101 cells are rejected by naive mice while A− 8101 tumor cells grow. To identify the antigen H-2Kb molecules were immunoprecipitated from A+ 8101 cells and peptides were eluted by acid. The sensitizing peptide was isolated by sequential reverse-phase HPLC and sequenced using microcapillary HPLC-triple quadruple mass spectrometry. The peptide, SNFVFAGI, matched the sequence of the DEAD box protein p68 RNA helicase except for a single amino acid substitution, caused by a single nucleotide change. This mutation was somatic since fibroblasts from the mouse of tumor origin expressed the wild-type sequence. The amino acid substitution created an anchor for binding of the mutant peptide to H-2Kb. Our results are consistent with mutant p68 being responsible for rejection of the tumor. Several functions of p68, which include nucleolar assembly and inhibition of DNA unwinding, may be mediated through its IQ domain, which was altered by the mutation. This is the first description of a somatic tumor–specific mutation in the coding region of a nucleic acid helicase. PMID:9034148

  19. Somatic reversion of some copia-like induced mutations, at the white locus of Drosophila melanogaster, after treatment with alkylating agents.

    PubMed

    Soriano, S; Creus, A; Marcos, R; Xamena, N

    1995-01-01

    It has been suggested that transposable elements can be associated with different types of genotoxic effects. For this reason it seems appropriate to outline suitable systems to detect changes in the phenotypic expression of the loci containing transposable elements, as well as those agents that induce such changes. The sex-linked white locus offers a suitable experimental system for studying such events because most of the spontaneous mutations at the white locus are the result of insertions of repeated mobile sequences, and it is easy to follow mutational changes of the locus due to the possibility of detecting even slight changes in eye color. Here we report the results obtained in different strains of Drosophila melanogaster with copia-like induced mutations at the white locus, after treatment with three alkylating agents: ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), and N-nitroso-N-ethylurea (ENU). The three insertional white mutants used in this work were wa4, wbf, and wsp55, with the wa2 mutation used as control because its mutant phenotype is the result of a point mutation instead of the insertion of a DNA fragment. Our data constitute evidence that EMS, MMS, and ENU induce a clear increase in the frequencies of somatic-revertant sectors in the three strains carrying a white allele with an inserted copia-like element. For the wa2 strain, whose mutant phenotype is the result of a point mutation, only ENU at the highest concentration tested is able to induce a significant increase in the somatic reversion frequency. In addition, our results indicate that the use of D. melanogaster strains with transposable elements in the white locus is suitable for detecting genotoxic damage induced by chemicals. PMID:7698106

  20. Somatic Cell-Induced Hyperacetylation, But Not Hypomethylation, Positively and Reversibly Affects the Efficiency of In Vitro Cloned Blastocyst Production in Cattle

    PubMed Central

    Jafarpour, Farnoosh; Hosseini, Sayed Morteza; Hajian, Mehdi; Forouzanfar, Mohsen; Ostadhosseini, Somayyeh; Abedi, Parvaneh; Gholami, Soghra; Ghaedi, Kamran; Gourabi, Hamid; Shahverdi, Abdol Hossein; Vosough, Ahmad Dizaj Taghi

    2011-01-01

    Abstract 5-Aza-2′-deoxycytidine (AzC), trichostatin A (TSA), and its natural mimetic, sodium butyrate (NaB), are antineoplastic drugs that can modify the epigenetic status of donor cells prior to somatic cell nuclear transfer (SCNT). In this study, we used fibroblast cells treated with these drugs to investigate the direct and indirect effects of induced changes in DNA methylation and acetylation of the lysine 9 residue of histone H3 (H3K9). Additionally, we assayed cellular characteristics (cell growth, cell proliferation, cell cycle progression, and apoptosis) and SCNT efficiency in response to these drugs as well as monitoring these effects 24 h after removing the drugs. We observed the following: (1) AzC, TSA, and NaB all showed dose-dependent effects on different cellular characteristics; (2) TSA and NaB induced H3K9 hyperacetylation accompanied by DNA hypermethylation, whereas AzC induced DNA hypomethylation with no effect on H3K9 hyperacetylation; (3) TSA and NaB improved cloning efficiency, whereas AzC reduced it; and (4) unlike AzC, the effects of TSA and NaB on cellular characteristics and SCNT efficiency were reversed following drug removal. Our results indicate that somatic cells treated with TSA and NaB show better survival and recovery rates following the removal of these drugs. Moreover, H3K9 hyperacetylation (induced with TSA and NaB), but not DNA hypomethylation (induced with AzC), favors cloning efficiency. PMID:21919704

  1. Integration-free reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) without viral vectors, recombinant DNA, and genetic modification.

    PubMed

    Heng, Boon Chin; Fussenegger, Martin

    2014-01-01

    Stem cells are envisaged to be integral components of multicellular systems engineered for therapeutic applications. The reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) via recombinant expression of a limited number of transcription factors, which was first achieved by Yamanaka and colleagues in 2007, heralded a major breakthrough in the stem cell field. Since then, there has been rapid progress in the field of iPSC generation, including the identification of various small molecules that can enhance reprogramming efficiency and reduce the number of different transcription factors required for reprogramming. Nevertheless, the major obstacles facing clinical applications of iPSCs are safety concerns associated with the use of viral vectors and recombinant DNA for expressing the appropriate transcription factors during reprogramming. In particular, permanent genetic modifications to newly reprogrammed iPSCs have to be avoided in order to meet stringent safety requirements for clinical therapy. These safety challenges can be overcome by new technology platforms that enable cellular reprogramming to iPSCs without the need to utilize either recombinant DNA or viral vectors. The use of recombinant cell-penetrating peptides and direct transfection of synthetic mRNA encoding appropriate transcription factors have both been shown to successfully reprogram somatic cells to iPSCs. It has also been shown more recently that the direct transfection of certain miRNA species can reprogram somatic cells to pluripotency without the need for any of the transcription factors commonly utilized for iPSC generation. This chapter describes protocols for iPSC generation with these new techniques, which would obviate the use of recombinant DNA and viral vectors in cellular reprogramming, thus avoiding permanent genetic modification to the reprogrammed cells.

  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. (Why) Does Evolution Favour Embryogenesis?

    PubMed

    Rensing, Stefan A

    2016-07-01

    Complex multicellular organisms typically possess life cycles in which zygotes (formed by gamete fusion) and meiosis occur. Canonical animal embryogenesis describes development from zygote to birth. It involves polarisation of the egg/zygote, asymmetric cell divisions, establishment of axes, symmetry breaking, formation of organs, and parental nutrition (at least in early stages). Similar developmental patterns have independently evolved in other eukaryotic lineages, including land plants and brown algae. The question arises whether embryo-like structures and associated developmental processes recurrently emerge because they are local optima of the evolutionary landscape. To understand which evolutionary principles govern complex multicellularity, we need to analyse why and how similar processes evolve convergently - von Baer's and Haeckel's phylotypic stage revisited in other phyla. PMID:26987708

  4. 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

  5. Effect of cotreatment of aspirin metabolites on mitomycin C-induced genotoxicity using the somatic mutation and recombination test in Drosophila melanogaster.

    PubMed

    Niikawa, Miki; Nakamura, Takeshi; Nagase, Hisamitsu

    2006-01-01

    In our previous reports, aspirin, an antipyretic analgesic, suppressed the genotoxicity of mitomycin C (MMC) in a somatic mutation and recombination test (SMART) in Drosophila melanogaster. In order to reveal the mechanism of the anti-genotoxicity of aspirin, we evaluated the suppressing ability of each aspirin metabolite, such as salicylic acid (SA), salicyluric acid (SUA), gentisic acid (GA), gentisuric acid (GUA), and 2,3-dihydroxybenzoic acid (DHBA), in SMART in Drosophila melanogaster using the cotreatment protocol in this report. SUA, GA, GUA, and DHBA reduced the number of the three types of spot induced by MMC without decrease of survival. These aspirin metabolites decreased the genotoxicity frequency of MMC for total spots in a dose-dependent manner. Furthermore, each metabolite decreased the genotoxicity frequency of MMC by approximately 80% at a dose of 40 mg/bottle, respectively. It is suggested that these metabolites are the main substances of anti-genotoxicity in the aspirin metabolic pathway. PMID:16931440

  6. [Relationship between epigenetics of sperm and embryogenesis].

    PubMed

    He, Yan-Fang; Ma, Jie-Hua; Pan, Lian-Jun; Huang, Yu-Feng

    2014-08-01

    Epigenetics comprises the modifications made in gene expressions without changing the DNA sequence itself. Significant epigenetic changes take place during spermatogenesis and fertilization and exert direct influences on embryogenesis. This article provides an overview of the latest researches on epigenetics of male germ cells and a brief discussion on the correlation of sperm with embryogenesis in four aspects: DNA methylation, histone modification, regulation of non-coding RNAs, and genomic imprinting. PMID:25195372

  7. Somatic mosaicism and disease.

    PubMed

    Frank, Steven A

    2014-06-16

    The large number of cell divisions required to make a human body inevitably leads to the accumulation of somatic mutations. Such mutations cause individuals to be somatic mosaics. Recent advances in genomic technology now allow measurement of somatic diversity. Initial studies confirmed the expected high levels of somatic mutations within individuals. Going forward, the big questions concern the degree to which those somatic mutations influence disease. Theory predicts that the frequency of mutant cells should vary greatly between individuals. Such somatic mutational variability between individuals could explain much of the diversity in the risk of disease. But how variable is mosaicism between individuals in reality? What is the relation between the fraction of cells carrying a predisposing mutation and the risk of disease? What kinds of heritable somatic change lead to disease besides classical DNA mutations? What molecular processes connect a predisposing somatic change to disease? We know that predisposing somatic mutations strongly influence the onset of cancer. Likewise, neurodegenerative diseases may often begin from somatically mutated cells. If so, both neurodegeneration and cancer may be diseases of later life for which much of the risk may be set by early life somatic mutations.

  8. 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.

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

    PubMed

    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

  10. Thidiazuron-induced morphogenetic response in petiole cultures of Pelargonium x hortorum and Pelargonium x domesticum and its histological analysis.

    PubMed

    Haensch, K-T

    2004-10-01

    The possibility of inducing somatic embryogenesis in petiole cultures of two cultivars of Pelargonium x hortorum and of one cultivar of Pelargonium x domesticum using thidiazuron (TDZ) was investigated. Petioles were cultivated on a modified Murashige and Skoog medium with different concentrations and application periods of TDZ. Regeneration was achieved with all TDZ treatments for all cultivars and was highly variable. Shoots of different shapes and somatic embryo-like structures were observed. Histological examination revealed that no somatic embryos were formed, and regenerants had to be classified as shoots and shoot-like or leaf-like structures. The importance of these results on the classification of regeneration induced by TDZ in these species and on the propagation of these pelargoniums is discussed.

  11. Bovine somatic cell nuclear transfer.

    PubMed

    Ross, Pablo J; Cibelli, Jose B

    2010-01-01

    Somatic cell nuclear transfer (SCNT) is a technique by which the nucleus of a differentiated cell is introduced into an oocyte from which its genetic material has been removed by a process called enucleation. In mammals, the reconstructed embryo is artificially induced to initiate embryonic development (activation). The oocyte turns the somatic cell nucleus into an embryonic nucleus. This process is called nuclear reprogramming and involves an important change of cell fate, by which the somatic cell nucleus becomes capable of generating all the cell types required for the formation of a new individual, including extraembryonic tissues. Therefore, after transfer of a cloned embryo to a surrogate mother, an offspring genetically identical to the animal from which the somatic cells where isolated, is born. Cloning by nuclear transfer has potential applications in agriculture and biomedicine, but is limited by low efficiency. Cattle were the second mammalian species to be cloned after Dolly the sheep, and it is probably the most widely used species for SCNT experiments. This is, in part due to the high availability of bovine oocytes and the relatively higher efficiency levels usually obtained in cattle. Given the wide utilization of this species for cloning, several alternatives to this basic protocol can be found in the literature. Here we describe a basic protocol for bovine SCNT currently being used in our laboratory, which is amenable for the use of the nuclear transplantation technique for research or commercial purposes. PMID:20336522

  12. Determination of genetic stability in long-term somatic embryogenic cultures and derived plantlets of cork oak using microsatellite markers.

    PubMed

    Lopes, Tina; Pinto, Glória; Loureiro, João; Costa, Armando; Santos, Conceição

    2006-09-01

    Microsatellites were used to test genetic stability in somatic embryos (SE) of Quercus suber L. The SE were obtained by a simple somatic embryogenesis protocol: leaf explants from two adult plants (QsG0, QsG5) and from two juvenile plants (QsGM1, QsGM2) were inoculated on Murashige and Skoog (MS) medium with 2,4-dichlorophenoxyacetic acid and zeatin. Calluses with primary embryogenic structures were transferred to MSWH (MS medium without growth regulators) and SE proliferated by secondary somatic embryogenesis. High morphological heterogeneity was found among cotyledonary SE. However, converted plants looked morphologically normal with well-developed rooting systems and shoots. The genetic stability of the plant material during the somatic embryogenesis process was evaluated by using six to eight nuclear microsatellites transferred from Q. myrsinifolia Blume, Q. petraea (Matts.) Liebl. and Q. robur L. Five of eight microsatellites distinguished among the genotypes analyzed, and for QsG0, QsGM1 and QsGM2, uniform microsatellite patterns were generally observed within and between SE and the respective donor genotypes. For genotype QsG5, the same pattern was observed in all samples analyzed except one, where the mutation percentage was 2.5%. We conclude that microsatellite markers can be used to assess genetic stability of clonal materials and to determine genetic stability throughout the process of somatic embryogenesis. The simple somatic embryogenesis protocol described has potential for the commercial propagation of Q. suber because it results in a low percentage of mutations.

  13. 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 ...

  14. Long-lived antigen-induced IgM plasma cells demonstrate somatic mutations and contribute to long-term protection

    PubMed Central

    Bohannon, Caitlin; Powers, Ryan; Satyabhama, Lakshmipriyadarshini; Cui, Ang; Tipton, Christopher; Michaeli, Miri; Skountzou, Ioanna; Mittler, Robert S.; Kleinstein, Steven H.; Mehr, Ramit; Lee, Frances Eun-Yun; Sanz, Ignacio; Jacob, Joshy

    2016-01-01

    Long-lived plasma cells are critical to humoral immunity as a lifelong source of protective antibodies. Antigen-activated B cells—with T-cell help—undergo affinity maturation within germinal centres and persist as long-lived IgG plasma cells in the bone marrow. Here we show that antigen-specific, induced IgM plasma cells also persist for a lifetime. Unlike long-lived IgG plasma cells, which develop in germinal centres and then home to the bone marrow, IgM plasma cells are primarily retained within the spleen and can develop even in the absence of germinal centres. Interestingly, their expressed IgV loci exhibit somatic mutations introduced by the activation-induced cytidine deaminase (AID). However, these IgM plasma cells are probably not antigen-selected, as replacement mutations are spread through the variable segment and not enriched within the CDRs. Finally, antibodies from long-lived IgM plasma cells provide protective host immunity against a lethal virus challenge. PMID:27270306

  15. Regulation of Specific Functions of Glial Cells in Somatic Hybrids, II. Control of Inducibility of Glycerol-3-Phosphate Dehydrogenase

    PubMed Central

    Davidson, Richard L.; Benda, Philippe

    1970-01-01

    Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) is induced when glial cells are exposed to hydrocortisone in vitro. In contrast, the enzyme activity in fibroblasts is not affected by the steroid. In an attempt to elucidate the mechanisms controlling inducibility, hybrids between glial cells and fibroblasts were studied. It was found that the activity of the enzyme does not increase when the hybrids are exposed to hydrocortisone. It was also shown that inducibility and the noninduced activity of enzyme are controlled independently. Comparisons of S-100 and glycerol phosphate dehydrogenase activity in the hybrids suggest that all the specialized functions characteristics of glial cells are not coordinately controlled. PMID:4321349

  16. Stress-related function of bHLH109 in somatic embryo induction in Arabidopsis.

    PubMed

    Nowak, Katarzyna; Gaj, Małgorzata D

    2016-04-01

    The bHLH109 gene of the bHLH family was identified among the transcription factor encoding genes that were differentially expressed in an embryogenic culture of Arabidopsis. A strong activation of bHLH109 expression was found to be associated with somatic embryogenesis (SE) induction. Several pieces of evidence suggested the involvement of bHLH109 in SE, including the high stimulation of the gene expression in SE-induced explants, which contrasts to the drastically lower level of the gene transcripts in the non-embryogenic callus and in tissue that is induced towards shoot regeneration via organogenesis. Moreover, in contrast to the overexpression of bHLH109, which has been indicated to enhance SE induction in a culture, the bhlh109 knock-out mutation was found to impair the embryogenic potential of explants. In order to identify the genes interacting with the bHLH109, the candidate co-expressed genes were identified in a yeast one hybrid assay. The in vitro regulatory interactions that were identified were verified through mutant and expression analysis. The results suggest that in SE bHLH109 acts as an activator of ECP63, a member of the LEA (LATE EMBRYOGENESIS ABUNDANT) family. Among the potential regulators of bHLH109, three candidates (At5g61620, bZIP4 and bZIP43) were indicated to possibly control bHLH109. The functions of all of the genes that are assumed to interact with bHLH109 are annotated to stress responses. Collectively, the results of the study provide new evidence that cell responses to stress that is imposed under in vitro conditions underlies the promotion of SE. bHLH109 may play a central role in the stress-related mechanism of SE induction via an increased accumulation of the LEA protein (ECP63), which results in the enhanced tolerance of the cells to stress. PMID:26973252

  17. Enolases: storage compounds in seeds? Evidence from a proteomic comparison of zygotic and somatic embryos of Cyclamen persicum Mill.

    PubMed

    Rode, Christina; Gallien, Sébastien; Heintz, Dimitri; Van Dorsselaer, Alain; Braun, Hans-Peter; Winkelmann, Traud

    2011-02-01

    Somatic embryogenesis is well established for the economic relevant ornamental crop Cyclamen and thus could supplement the elaborate propagation via seeds. However, the use of somatic embryogenesis for commercial large scale propagation is still limited due to physiological disorders and asynchronous development within emerged embryos. To overcome these problems, profound knowledge of the physiological processes in Cyclamen embryogenesis is essential. Thus, the proteomes of somatic and zygotic embryos were characterised in a comparative approach. Protein separation via two dimensional IEF-SDS PAGE led to a resolution of more than 1,000 protein spots/gel. Overall, 246 proteins were of differential abundance in the two tissues compared. Mass spectrometry analysis of the 300 most abundant protein spots resulted in the identification of 247 proteins, which represent 90 distinct protein species. Fifty-five percent of the 247 proteins belong to only three physiological categories: glycolysis, protein folding and stress response. The latter physiological process was especially predominant in the somatic embryos. Remarkably, the glycolytic enzyme enolase was the protein most frequently detected and thus is supposed to play an important role in Cyclamen embryogenesis. Data are presented that indicate involvement of "small enolases" as storage proteins in Cyclamen. A digital reference map was established via a novel software tool for the web-based presentation of proteome data linked to KEGG and ExPasy protein-databases and both were made publicly available online.

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

    PubMed

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

    2015-01-15

    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.

  19. 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…

  20. Cognitive and somatic anxiety.

    PubMed

    Steptoe, A; Kearsley, N

    1990-01-01

    Three hundred and forty adults (including sports players, recreational exercisers, mediators and sedentary controls) completed three inventories purporting to measure cognitive and somatic aspects of anxiety. These were the Cognitive-Somatic Anxiety Questionnaire (CSAQ) devised by Schwartz, Davidson & Goleman (Psychosomatic Medicine, 40, 321-328, 1978), the Worry-Emotionality Scale (WES, Morris, Davis & Hutchens, Journal of Educational Psychology, 73, 541-555, 1981) and the Lehrer-Woolfolk (1982) Anxiety Symptom Questionnaire (LWASQ). Factor analysis of the CSAQ and WES identified distinct cognitive and somatic anxiety factors in both inventories. Higher somatic than cognitive ratings were recorded on the CSAQ and WES, while the pattern was reversed on the LWASQ. The CSAQ can tentatively be recommended as a useful measure of these two anxiety components. We were unable to confirm an observation made previously in the literature that practice of meditation is associated with reduced cognitive anxiety, or that exercise is linked with lower somatic anxiety.

  1. Protective effects of new medicinal mushroom, Grifola gargal singer (higher Basidiomycetes), on induced DNA damage in somatic cells of Drosophila melanogaster.

    PubMed

    Postemsky, Pablo Daniel; Palermo, Ana Maria; Curvetto, Néstor Raúl

    2011-01-01

    Grifola gargal is an edible mushroom with attributed antioxidant properties. Different sources of G. gargal materials, i.e., fruit bodies and mycelia grown in liquid or solid media, were used to study its potential protective capacity when somatic mutation and recombination is induced in Drosophila melanogaster using DMBA (7-12-dimethyl-benz(α)anthracene) as promutagen. Heterozygote larvae (white/white+) were grown in media with different concentrations of DMBA. Grifola gargal fruit bodies (GgFB) or mycelia from liquid culture (GgLC) or from solid culture (GgWG), i.e., biotransformed wheat kernel flour, were added to the culture media in combined treatments with DMBA. Water, DMBA solvent, or wheat flour (WF) plus DMBA solvent were used as negative controls. Larval mortality increased from 9% to 11% in negative controls to 31% to 36% in DMBA treatments. The addition of GgFB, GgLC, or GgWG materials produced a protective effect on 25 μmol/vial DMBA-induced mortality. Mutations observed in SMART, as light spots per 100 eyes (LS/100 eyes), increased with increasing doses of DMBA; this was also true when considering the mutation incidence expressed as percentage of eyes exhibiting light spots (% eyes with LS). Interestingly, mycelia from GgFB, GgLC, or GgWG, in the presence of 25 μmol/vial DMBA, showed lower values in SMART of both the total LS/100 eyes and the percentage of eyes with LS. Thus, Grifola gargal materials were not only nontoxic, but in combination with 25 μmol/vial DMBA lowered the mortality induced by the promutagen and showed antimutagenic effects. Protective effects of G. gargal against DMBA are discussed in terms of the onset of desmutagenic and/or bioantimutagenic mechanisms of detoxification in the host organism, probably due to some bioactive compounds known to occur in higher mushrooms. PMID:22181846

  2. Histological and transcript analyses of intact somatic embryos in an elite maize (Zea mays L.) inbred line Y423.

    PubMed

    Liu, Beibei; Su, Shengzhong; Wu, Ying; Li, Ying; Shan, Xiaohui; Li, Shipeng; Liu, Hongkui; Dong, Haixiao; Ding, Meiqi; Han, Junyou; Yuan, Yaping

    2015-07-01

    Intact somatic embryos were obtained from an elite maize inbred line Y423, bred in our laboratory. Using 13-day immature embryos after self-pollination as explants, and after 4-5 times subculture, a large number of somatic embryos were detected on the surface of the embryonic calli on the medium. The intact somatic embryos were transferred into the differential medium, where the plantlets regenerated with shoots and roots forming simultaneously. Histological analysis and scanning electron micrographs confirmed the different developmental stages of somatic embryogenesis, including globular-shaped embryo, pear-shaped embryo, scutiform embryo, and mature embryo. cDNA-amplified fragment length polymorphism (cDNA-AFLP) was used for comparative transcript profiling between embryogenic and non-embryogenic calli of a new elite maize inbred line Y423 during somatic embryogenesis. Differentially expressed genes were cloned and sequenced. Gene Ontology analysis of 117 candidate genes indicated their involvement in cellular component, biological process and molecular function. Nine of the candidate genes were selected. The changes in their expression levels during embryo induction and regeneration were analyzed in detail using quantitative real-time PCR. Two full-length cDNA sequences, encoding ZmSUF4 (suppressor of fir 4-like protein) and ZmDRP3A (dynamin-related protein), were cloned successfully from intact somatic embryos of the elite inbred maize line Y423. Here, a procedure for maize plant regeneration from somatic embryos is described. Additionally, the possible roles of some of these genes during the somatic embryogenesis has been discussed. This study is a systematic analysis of the cellular and molecular mechanism during the formation of intact somatic embryos in maize.

  3. Prolonged interval between fusion and activation impairs embryonic development by inducing chromosome scattering and nuclear aneuploidy in pig somatic cell nuclear transfer.

    PubMed

    You, Jinyoung; Song, Kilyoung; Lee, Eunsong

    2010-01-01

    The aim of the present study was to examine the effect of various intervals between electrofusion and activation (FA interval) on the nuclear remodelling and development of somatic cell nuclear transfer (SCNT) embryos in pigs. Reconstructed oocytes were activated at 0 (simultaneous fusion and activation; SFA), 1, 2 and 3 h (delayed activation) after electrofusion; these groups were designated as DA1, DA2 and DA3, respectively. When oocyte nuclear status was examined at 0.5, 1, 2 and 3 h after electrofusion, the incidence of chromosome scattering was increased (P < 0.01) as the FA interval was extended (0.0%, 12.0%, 77.3% and 78.0%, respectively). Extending the FA interval led to an increase (P < 0.01) in the percentage of oocytes containing multiple (>or=3) pseudopronuclei (PPN) (0.0% of SFA; 5.3% of DA1; 21.7% of DA2; and 33.5% of DA3). The development of SCNT embryos to the blastocyst stage was decreased (P < 0.05) in DA2 (5.7%) and DA3 (5.0%) compared with SFA (18.1%) and DA1 (19.5%). Our results demonstrate that extending the FA interval impairs the development of SCNT pig embryos by inducing chromosome scattering and the formation of multiple PPN, which may result in increased nuclear aneuploidy.

  4. Rapid and efficient reprogramming of somatic cells to induced pluripotent stem cells by retinoic acid receptor gamma and liver receptor homolog 1

    PubMed Central

    Wang, Wei; Yang, Jian; Liu, Hui; Lu, Dong; Chen, Xiongfeng; Zenonos, Zenon; Campos, Lia S.; Rad, Roland; Guo, Ge; Zhang, Shujun; Bradley, Allan; Liu, Pentao

    2011-01-01

    Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by expressing four transcription factors: Oct4, Sox2, Klf4, and c-Myc. Here we report that enhancing RA signaling by expressing RA receptors (RARs) or by RA agonists profoundly promoted reprogramming, but inhibiting it using a RAR-α dominant-negative form completely blocked it. Coexpressing Rarg (RAR-γ) and Lrh-1 (liver receptor homologue 1; Nr5a2) with the four factors greatly accelerated reprogramming so that reprogramming of mouse embryonic fibroblast cells to ground-state iPSCs requires only 4 d induction of these six factors. The six-factor combination readily reprogrammed primary human neonatal and adult fibroblast cells to exogenous factor-independent iPSCs, which resembled ground-state mouse ES cells in growth properties, gene expression, and signaling dependency. Our findings demonstrate that signaling through RARs has critical roles in molecular reprogramming and that the synergistic interaction between Rarg and Lrh1 directs reprogramming toward ground-state pluripotency. The human iPSCs described here should facilitate functional analysis of the human genome. PMID:21990348

  5. 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

  6. Microspore-derived embryogenesis in pepper (Capsicum annuum L.): subcellular rearrangements through development.

    PubMed

    Bárány, Ivett; González-Melendi, Pablo; Fadón, Begoña; Mitykó, Judit; Risueño, María C; Testillano, Pilar S

    2005-09-01

    Background information. In vitro-cultured microspores, after an appropriate stress treatment, can switch towards an embryogenic pathway. This process, known as microspore embryogenesis, is an important tool in plant breeding. Basic studies on this process in economically interesting crops, especially in recalcitrant plants, are very limited and the sequence of events is poorly understood. In situ studies are very convenient for an appropriate dissection of microspore embryogenesis, a process in which a mixture of different cell populations (induced and non-induced) develop asynchronically.Results. In the present study, the occurrence of defined subcellular rearrangements has been investigated during early microspore embryogenesis in pepper, an horticultural crop of agronomic interest, in relation to proliferation and differentiation events. Haploid plants of Capsicum annuum L. (var. Yolo Wonder B) have been regenerated from in vitro anther cultures by a heat treatment at 35 degrees C for 8 days. Morphogenesis of microspore-derived embryos has been analysed, at both light and electron microscopy levels, using low-temperature-processed, well-preserved specimens. The comparison with the normal gametophytic development revealed changes in cell organization after embryogenesis induction, and permitted the characterization of the time sequence of a set of structural events, not previously defined in pepper, related to the activation of proliferative activity and differentiation. These changes mainly affected the plastids, the vacuolar compartment, the cell wall and the nucleus. Further differentiation processes mimicked that of the zygotic development.Conclusions. The reported changes can be considered as markers of the microspore embryogenesis. They have increased the understanding of the mechanisms controlling the switch and progression of the microspore embryogenesis, which could help to improve its efficiency and to direct strategies, especially in agronomically

  7. Early Zebrafish Embryogenesis Is Susceptible to Developmental TDCPP Exposure

    PubMed Central

    McGee, Sean P.; Cooper, Ellen M.; Stapleton, Heather M.

    2012-01-01

    Background: Chlorinated phosphate esters (CPEs) are widely used as additive flame retardants for low-density polyurethane foams and have frequently been detected at elevated concentrations within indoor environmental media. Objectives: To begin characterizing the potential toxicity of CPEs on early vertebrate development, we examined the developmental toxicity of four CPEs used in polyurethane foam: tris(1,3-dichloro-2-propyl) phosphate (TDCPP), tris(2-chloroethyl) phosphate (TCEP), tris(1-chloro-2-propyl) phosphate (TCPP), and 2,2-bis(chloromethyl)propane-1,3-diyl tetrakis(2-chlorethyl) bis(phosphate) (V6). Methods: Using zebrafish as a model for vertebrate embryogenesis, we first screened the potential teratogenic effects of TDCPP, TCEP, TCPP, and V6 using a developmental toxicity assay. Based on these results, we focused on identification of susceptible windows of developmental TDCPP exposure as well as evaluation of uptake and elimination of TDCPP and bis(1,3-dichloro-2-propyl)phosphate (BDCPP, the primary metabolite) within whole embryos. Finally, because TDCPP-specific genotoxicity assays have, for the most part, been negative in vivo and because zygotic genome remethylation is a key biological event during cleavage, we investigated whether TDCPP altered the status of zygotic genome methylation during early zebrafish embryogenesis. Results: Overall, our findings suggest that the cleavage period during zebrafish embryogenesis is susceptible to TDCPP-induced delays in remethylation of the zygotic genome, a mechanism that may be associated with enhanced developmental toxicity following initiation of TDCPP exposure at the start of cleavage. Conclusions: Our results suggest that further research is needed to better understand the effects of a widely used and detected CPE within susceptible windows of early vertebrate development. PMID:23017583

  8. Protective effect of l-carnitine against acrylamide-induced DNA damage in somatic and germ cells of mice

    PubMed Central

    Alzahrani, Hind Abdullah Seed

    2010-01-01

    Recent findings of acrylamide (AA) in many common foods have sparked renewed interest in assessing human health hazards. AA was evaluated by the International Agency for Research on Cancer as probably carcinogenic to humans. For this reason, the aim of this study is to evaluate the potential genotoxic effect of AA using chromosomal aberration analysis and micronucleus (MN) test in mouse bone-marrow cells and morphological sperm abnormalities. The result of the present work indicated that treatment with a single dose of 10, 20, or 30 mg/kg b.wt. of AA for 24 h and the repeated dose of 10 mg/kg b.wt. for 1and 2 weeks induced a statistically significant increase in the percentage of chromosomal aberrations and micronuclei in bone- marrow cells. These percentages reduced significantly in all groups treated with AA and the protective agent l-carnitine. Also the results indicated that the dose 10, 20 and 30 mg/kg b.wt. of AA induced a statistically significant percentage of morphological sperm abnormalities compared with the control group. Such effect reached its maximum (7.24 ± 0.61) with the highest tested dose which reduced to (4.02 ± 0.58) in the group treated with the same dose of AA and l-carnitine. In conclusion, the results confirm the protective role of LC against the mutagenicity of AA. PMID:23961101

  9. Somatization and conversion disorder.

    PubMed

    Hurwitz, Trevor A

    2004-03-01

    Somatization is the psychological mechanism whereby psychological distress is expressed in the form of physical symptoms. The psychological distress in somatization is most commonly caused by a mood disorder that threatens mental stability. Conversion disorder occurs when the somatic presentation involves any aspect of the central nervous system over which voluntary control is exercised. Conversion reactions represent fixed ideas about neurologic malfunction that are consciously enacted, resulting in psychogenic neurologic deficits. Treatment is complex and lengthy; it includes recovery of neurologic function aided by narcoanalysis and identification and treatment of the primary psychiatric disorder, usually a mood disorder. PMID:15101499

  10. Somatic hybrid plants from sexually incompatible woody species: Citrus reticulata and Citropsis gilletiana.

    PubMed

    Grosser, J W; Gmitter, F G; Tusa, N; Chandler, J L

    1990-04-01

    Allotetraploid intergeneric somatic hybrid plants between Citrus reticulata Blanco cv. Cleopatra mandarin and Citropsis gilletiana Swing. & M. Kell. (common name Gillet's cherry orange) were regenerated following protoplast fusion. Cleopatra protoplasts were isolated from an ovule-derived embryogenic suspension culture and fused chemically with leaf-derived protoplasts of Citropsis gilletiana. Cleopatra mandarin and somatic hybrid plants were regenerated via somatic embryogenesis. Hybrid plant identification was based on differential leaf morphology, root-tip cell chromosome number, and electrophoretic analyses of phosphoglucose mutase (PGM) and phosphohexose isomerase (PHI) isozyme banding patterns. This is the first somatic hybrid within the Rutaceae reported that does not have Citrus sinensis (sweet orange) as a parent, and the first produced with a commercially important citrus rootstock and a complementary but sexually incompatible, related species.

  11. Ethanol induced attenuation of oxidative stress is unable to alter mRNA expression pattern of catalase, glutathione reductase, glutathione-S- transferase (GST1A), and superoxide dismutase (SOD3) enzymes in Japanese rice fish (Oryzias latipes) embryogenesis

    PubMed Central

    Wu, Minghui; Shariat-Madar, Bahbak; Haron, Mona H.; Wu, Mengmeng; Khan, Ikhlas A.; Dasmahapatra, Asok K.

    2010-01-01

    Although the mechanism of ethanol toxicity during embryogenesis is unknown, our earlier studies on Japanese rice fish (Oryzias latipes) embryos indicated that the effects might be mediated through oxidative stress. In this study we have determined the oxidative stress and the mRNA content of four antioxidant enzymes (catalase, glutathione reductase, glutathione-S-transferase, and superoxide dismutase) during Japanese rice fish embryogenesis (from 0 day post-fertilization to hatching) and after exposing the embryos to ethanol (100 and 300 mM) for 48 h at three stages (0–2, 1–3 and 4–6 day post fertilization, dpf) of organogenesis. We observed that oxidative stress was minimal in blastula, gastrula or neurula stages, increased gradually with the advancement of morphogenesis and reached its maximum level in hatchlings. The antioxidant enzyme mRNAs were constitutively expressed throughout development; however, the expression pattern was not identical among the enzymes. Catalase and superoxide dismutase (SOD) mRNAs were minimal in the fertilized eggs, but increased significantly in 1 dpf and then either sharply dropped (SOD) or maintained a steady-state (catalase). Glutathione S-transferase (GST) was very high in fertilized eggs and sharply dropped 1 dpf and then gradually increased thereafter. Glutathione reductase (GR) maintained a steady-state throughout the development. Ethanol was able to attenuate oxidative stress in embryos exposed only to 300 mM 1–3 dpf; no significant difference with controls was observed in other ethanol-treated groups. The antioxidant enzyme mRNAs also remained unaltered after ethanol treatment. From these data we conclude that the attenuation of oxidative stress by ethanol is probably due to the inhibition of normal growth of the embryos rather than by inhibiting catalase, GST, GR or SOD- dependent activities. PMID:20965276

  12. 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

  13. Wild worm embryogenesis harbors ubiquitous polygenic modifier variation

    PubMed Central

    Paaby, Annalise B; White, Amelia G; Riccardi, David D; Gunsalus, Kristin C; Piano, Fabio; Rockman, Matthew V

    2015-01-01

    Embryogenesis is an essential and stereotypic process that nevertheless evolves among species. Its essentiality may favor the accumulation of cryptic genetic variation (CGV) that has no effect in the wild-type but that enhances or suppresses the effects of rare disruptions to gene function. Here, we adapted a classical modifier screen to interrogate the alleles segregating in natural populations of Caenorhabditis elegans: we induced gene knockdowns and used quantitative genetic methodology to examine how segregating variants modify the penetrance of embryonic lethality. Each perturbation revealed CGV, indicating that wild-type genomes harbor myriad genetic modifiers that may have little effect individually but which in aggregate can dramatically influence penetrance. Phenotypes were mediated by many modifiers, indicating high polygenicity, but the alleles tend to act very specifically, indicating low pleiotropy. Our findings demonstrate the extent of conditional functionality in complex trait architecture. DOI: http://dx.doi.org/10.7554/eLife.09178.001 PMID:26297805

  14. 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...

  15. 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...

  16. Metabolite profiling reveals clear metabolic changes during somatic embryo development of Norway spruce (Picea abies).

    PubMed

    Businge, Edward; Brackmann, Klaus; Moritz, Thomas; Egertsdotter, Ulrika

    2012-02-01

    Progress on industrial-scale propagation of conifers by somatic embryogenesis has been hampered by the differences in developmental capabilities between cell lines, which are limiting the capture of genetic gains from breeding programs. In this study, we investigated the metabolic events occurring during somatic embryo development in Norway spruce to establish a better understanding of the fundamental metabolic events required for somatic embryo development. Three embryogenic cell lines of Norway spruce (Picea abies (L.) Karst) with different developmental capabilities were studied during somatic embryo development from proliferation of proembryogenic masses to mature somatic embryos. The three different cell lines displayed normal, aberrant and blocked somatic embryo development. Metabolite profiles from four development stages in each of the cell lines were obtained using combined gas chromatography-mass spectrometry. Multivariate discriminant analyses of the metabolic data revealed significant metabolites (P  ≤  0.05) for each development stage and transition. The results suggest that endogenous auxin and sugar signaling affects initial stages of somatic embryo development. Furthermore, the results highlight the importance of a timed stress response and the presence of stimulatory metabolites during late stages of embryo development.

  17. 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

  18. 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…

  19. 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.

  20. 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

  1. 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.

  2. 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.

  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. Specification of the somatic musculature in Drosophila.

    PubMed

    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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. Somatic reduction in cycads.

    PubMed

    Storey, W B

    1968-02-01

    Recurrent somatic reduction is a normal ontogenetic process in apogeotropic roots of cycads, which develop into dichotomously branching coralloid masses. The reduced cells make up part of a ring of differentiated cortical tissue lying midway between the pericycle and the epidermis; they serve as fillers among the large cells and become charged with slime. The differentiated tissue is colonized by a species of blue-green algae.

  10. Gamete derivation from embryonic stem cells, induced pluripotent stem cells or somatic cell nuclear transfer-derived embryonic stem cells: state of the art

    PubMed Central

    Easley, Charles A.; Simerly, Calvin R.; Schatten, Gerald

    2015-01-01

    Generating gametes from pluripotent stem cells (PSCs) has many scientific justifications and several biomedical rationales. Here, we consider several strategies for deriving gametes from PSCs from mice and primates (human and non-human) and their anticipated strengths, challenges and limitations. Although the ‘Weismann barrier’, which separates the mortal somatic cell lineages from the potentially immortal germline, has long existed, breakthroughs first in mice and now in humans are artificially creating germ cells from somatic cells. Spermatozoa with full reproductive viability establishing multiple generations of seemingly normal offspring have been reported in mice and, in humans, haploid spermatids with correct parent-of-origin imprints have been obtained. Similar progress with making oocytes has been published using mouse PSCs differentiated in vitro into primordial germ cells, which are then cultured after xenografting reconstructed artificial ovaries. Progress in making human oocytes artificially is proving challenging. The usefulness of these artificial gametes, from assessing environmental exposure toxicity to optimising medical treatments to prevent negative off-target effects on fertility, may prove invaluable, as may basic discoveries on the fundamental mechanisms of gametogenesis. PMID:25472048

  11. Somatic responses in behavioral inhibition.

    PubMed

    Whitney, Paul; Hinson, John M; Wirick, Aaron; Holben, Heather

    2007-03-01

    In the present study, skin conductance responses (SCRs) were measured postdecision and prefeedback in a go/no-go (GNG) task in which participants used response feedback to learn when to respond or not to respond to numeric stimuli. Like somatic markers in gambling tasks and somatic reactions to error monitoring in choice reaction time tasks, SCR patterns distinguished between correct and incorrect trials over time. These somatic reactions were disrupted by a reversal of GNG contingencies, and they were facilitated by pretraining of the stimulus-response mappings. In all cases, however, the somatic reactions appeared to be a product of competent decision making rather than a contributor to performance. Differential somatic responses to good and bad choices appear to be a robust and fairly general phenomenon, but researchers should be cautious in assuming that the somatic responses contribute to performance.

  12. 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

  13. Ectopic expression of the Brassica SHOOTMERISTEMLESS attenuates the deleterious effects of the auxin transport inhibitor TIBA on somatic embryo number and morphology.

    PubMed

    Elhiti, Mohamed; Stasolla, Claudio

    2011-02-01

    The auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) is a useful compound for investigating the role of auxin flow during plant growth and development. In Arabidopsis lines, applications of TIBA during the induction phase of somatic embryogenesis inhibit embryo development and induce the differentiation of the meristematic cells of the shoot apical meristem (SAM), leading to the fusion of the cotyledons. These abnormalities were associated to changes in the expression levels of auxin transporter genes (PINs) and endogenous distribution of IAA. Treatments of TIBA caused a rapid accumulation of IAA within the epidermal and cortical root cells of the explants (bent-cotyledon zygotic embryos), as well as in the apical and sub-apical cells of the callus generated by the surface of the cotyledons of the explants. Within the callus only a few cells acquired meristematic characteristics, and this was associated to low expression levels of genes involved in embryogenic cell fate acquisition, such as WUSCHEL (WUS), LEAFY COTYLEDON 1 and 2. All these deleterious effects were attenuated when TIBA was administered to lines over-expressing SHOOT MERISTEMLESS (STM) isolated from Brassica oleracea (Bo), B. napus (Bn), and B. rapa (Br). Of interest, TIBA-treated explants of Arabidopsis lines over-expressing the Brassica STM were able to produce a large number of embryogenic cells and somatic embryos which exhibited a normal morphology and two distinct cotyledons. A proposed reason for this behaviour was ascribed to the ability of the transformed tissue to retain a normal distribution of auxin in the presence of TIBA. Proper localization of auxin might be required for the normal expression of several genes needed for the acquisition of embryogenic competence and formation of somatic embryos.

  14. Ectopic expression of the Brassica SHOOTMERISTEMLESS attenuates the deleterious effects of the auxin transport inhibitor TIBA on somatic embryo number and morphology.

    PubMed

    Elhiti, Mohamed; Stasolla, Claudio

    2011-02-01

    The auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) is a useful compound for investigating the role of auxin flow during plant growth and development. In Arabidopsis lines, applications of TIBA during the induction phase of somatic embryogenesis inhibit embryo development and induce the differentiation of the meristematic cells of the shoot apical meristem (SAM), leading to the fusion of the cotyledons. These abnormalities were associated to changes in the expression levels of auxin transporter genes (PINs) and endogenous distribution of IAA. Treatments of TIBA caused a rapid accumulation of IAA within the epidermal and cortical root cells of the explants (bent-cotyledon zygotic embryos), as well as in the apical and sub-apical cells of the callus generated by the surface of the cotyledons of the explants. Within the callus only a few cells acquired meristematic characteristics, and this was associated to low expression levels of genes involved in embryogenic cell fate acquisition, such as WUSCHEL (WUS), LEAFY COTYLEDON 1 and 2. All these deleterious effects were attenuated when TIBA was administered to lines over-expressing SHOOT MERISTEMLESS (STM) isolated from Brassica oleracea (Bo), B. napus (Bn), and B. rapa (Br). Of interest, TIBA-treated explants of Arabidopsis lines over-expressing the Brassica STM were able to produce a large number of embryogenic cells and somatic embryos which exhibited a normal morphology and two distinct cotyledons. A proposed reason for this behaviour was ascribed to the ability of the transformed tissue to retain a normal distribution of auxin in the presence of TIBA. Proper localization of auxin might be required for the normal expression of several genes needed for the acquisition of embryogenic competence and formation of somatic embryos. PMID:21421384

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. The p53-induced lincRNA-p21 derails somatic cell reprogramming by sustaining H3K9me3 and CpG methylation at pluripotency gene promoters.

    PubMed

    Bao, Xichen; Wu, Haitao; Zhu, Xihua; Guo, Xiangpeng; Hutchins, Andrew P; Luo, Zhiwei; Song, Hong; Chen, Yongqiang; Lai, Keyu; Yin, Menghui; Xu, Lingxiao; Zhou, Liang; Chen, Jiekai; Wang, Dongye; Qin, Baoming; Frampton, Jon; Tse, Hung-Fat; Pei, Duanqing; Wang, Huating; Zhang, Biliang; Esteban, Miguel A

    2015-01-01

    Recent studies have boosted our understanding of long noncoding RNAs (lncRNAs) in numerous biological processes, but few have examined their roles in somatic cell reprogramming. Through expression profiling and functional screening, we have identified that the large intergenic noncoding RNA p21 (lincRNA-p21) impairs reprogramming. Notably, lincRNA-p21 is induced by p53 but does not promote apoptosis or cell senescence in reprogramming. Instead, lincRNA-p21 associates with the H3K9 methyltransferase SETDB1 and the maintenance DNA methyltransferase DNMT1, which is facilitated by the RNA-binding protein HNRNPK. Consequently, lincRNA-p21 prevents reprogramming by sustaining H3K9me3 and/or CpG methylation at pluripotency gene promoters. Our results provide insight into the role of lncRNAs in reprogramming and establish a novel link between p53 and heterochromatin regulation.

  20. 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

    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

  1. 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

  2. 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.

  3. 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

  4. Estrogen receptors bind to and activate the HOXC4/HoxC4 promoter to potentiate HoxC4-mediated activation-induced cytosine deaminase induction, immunoglobulin class switch DNA recombination, and somatic hypermutation.

    PubMed

    Mai, Thach; Zan, Hong; Zhang, Jinsong; Hawkins, J Seth; Xu, Zhenming; Casali, Paolo

    2010-11-26

    Estrogen enhances antibody and autoantibody responses through yet to be defined mechanisms. It has been suggested that estrogen up-regulates the expression of activation-induced cytosine deaminase (AID), which is critical for antibody class switch DNA recombination (CSR) and somatic hypermutation (SHM), through direct activation of this gene. AID, as we have shown, is induced by the HoxC4 homeodomain transcription factor, which binds to a conserved HoxC4/Oct site in the AICDA/Aicda promoter. Here we show that estrogen-estrogen receptor (ER) complexes do not directly activate the AID gene promoter in B cells undergoing CSR. Rather, they bind to three evolutionarily conserved and cooperative estrogen response elements (EREs) we identified in the HOXC4/HoxC4 promoter. By binding to these EREs, ERs synergized with CD154 or LPS and IL-4 signaling to up-regulate HoxC4 expression, thereby inducing AID and CSR without affecting B cell proliferation or plasmacytoid differentiation. Estrogen administration in vivo significantly potentiated CSR and SHM in the specific antibody response to the 4-hydroxy-3-nitrophenylacetyl hapten conjugated with chicken γ-globulin. Ablation of HoxC4 (HoxC4(-/-)) abrogated the estrogen-mediated enhancement of AID gene expression and decreased CSR and SHM. Thus, estrogen enhances AID expression by activating the HOXC4/HoxC4 promoter and inducing the critical AID gene activator, HoxC4.

  5. 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

  6. A partially disarmed vir helper plasmid, pKYRT1, in conjunction with 2,4-dichlorophenoxyactic acid promotes emergence of regenerable transgenic somatic embryos from immature cotyledons of soybean.

    PubMed

    Ko, Tae-Seok; Lee, Sangman; Farrand, Stephen K; Korban, Schuyler S

    2004-02-01

    Agrobacterium tumefaciens strain KYRT1 harboring the virulence helper plasmid pKYRT1 induces transgenic somatic embryos (SEs) at high frequency from infected immature soybean cotyledons. KYRT1 is derived from the highly oncogenic strain Chry5. However, pKYRT1 is not completely disarmed and still contains an entire T-right (T(R)) and a portion of T-left (T(L)). In this report, binary strains, each carrying fully disarmed vir helper plasmids including pKPSF2, which is a fully disarmed version of pKYRT1, were compared to strain KYRT1 for their ability to induce transgenic SEs on immature cotyledons of soybean. Six weeks following cocultivation, histochemical GUS assays of cultured explants indicated that all fully disarmed vir helper plasmids transferred their binary T-DNA, containing a GUS-intron gene, into soybean tissues. However, none of these transformed tissues developed SEs on medium with or without 2,4-dichlorophenoxyactic acid (2,4-D). On the other hand, immature cotyledons cocultivated with strain KYRT1 exhibited high induction of transgenic SEs, but only on medium supplemented with 2,4-D. Derivatives of strain Chry5 harboring other vir helper plasmids did not induce transgenic SEs under any conditions tested, thus suggesting that the chromosomal background of KYRT1 alone was not sufficient to promote somatic embryogenesis. PCR analysis indicated that 55% of transgenic embryogenic cultures and 29% of transgenic T(0) soybean plants derived by transformation using strain KYRT1 contained T(R) from pKYRT1 in addition to the uidA gene from the binary construct. None of the transgenic tissues or T(0) plants contained T(L) DNA. These results suggest that some function coded for by T(R) of pKYRT1 influences somatic embryogenesis in conjunction with exposure of the plant tissues to 2,4-D. Since the co-transformation frequency of the undesirable T-DNA sequences from the vir helper plasmid was relatively low, the partially disarmed strain KYRT1 will likely be very

  7. 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.

  8. Acrolein and embryogenesis: an experimental study

    SciTech Connect

    Chhibber, G.; Cilani, S.H.

    1986-01-01

    The effects of acrolein were studied on the chick embryos of 48 and 72 hr of incubation. Acrolein was dissolved in physiological saline and injected into the air sacs of the eggs at doses ranging from 0.001 to 0.1 mg per egg. The controls received and equal amount of saline only (0.1 ml per egg). All the embryos including controls were examined at Day 13. In all, 600 eggs were utilized for this investigation. At 48 hr incubation, the percentage survival ranged from 80 to 0 as the dosage of acrolein was increased. Embryonic mortality following 72 hr incubation did not increase significantly at any dose level. Gross malformations such as short and twisted limbs, everted viscera, microphthalmia, short and twisted neck, and hemorrhage over the body were observed. The frequency and the types of gross abnormalities did not vary much in the 48- or 72-hr-treated groups. The incidence of malformation in the controls was low. The results of this study indicates that acrolein is embryotoxic at higher doses and moderately teratogenic to chick embryogenesis.

  9. Metabolome Analysis of Drosophila melanogaster during Embryogenesis

    PubMed Central

    An, Phan Nguyen Thuy; Yamaguchi, Masamitsu; Bamba, Takeshi; Fukusaki, Eiichiro

    2014-01-01

    The Drosophila melanogaster embryo has been widely utilized as a model for genetics and developmental biology due to its small size, short generation time, and large brood size. Information on embryonic metabolism during developmental progression is important for further understanding the mechanisms of Drosophila embryogenesis. Therefore, the aim of this study is to assess the changes in embryos’ metabolome that occur at different stages of the Drosophila embryonic development. Time course samples of Drosophila embryos were subjected to GC/MS-based metabolome analysis for profiling of low molecular weight hydrophilic metabolites, including sugars, amino acids, and organic acids. The results showed that the metabolic profiles of Drosophila embryo varied during the course of development and there was a strong correlation between the metabolome and different embryonic stages. Using the metabolome information, we were able to establish a prediction model for developmental stages of embryos starting from their high-resolution quantitative metabolite composition. Among the important metabolites revealed from our model, we suggest that different amino acids appear to play distinct roles in different developmental stages and an appropriate balance in trehalose-glucose ratio is crucial to supply the carbohydrate source for the development of Drosophila embryo. PMID:25121768

  10. Reprogramming of Somatic Cells Towards Pluripotency by Cell Fusion.

    PubMed

    Malinowski, Andrzej R; Fisher, Amanda G

    2016-01-01

    Pluripotent reprogramming can be dominantly induced in a somatic nucleus upon fusion with a pluripotent cell such as embryonic stem (ES) cell. Cell fusion between ES cells and somatic cells results in the formation of heterokaryons, in which the somatic nuclei begin to acquire features of the pluripotent partner. The generation of interspecies heterokaryons between mouse ES- and human somatic cells allows an experimenter to distinguish the nuclear events occurring specifically within the reprogrammed nucleus. Therefore, cell fusion provides a simple and rapid approach to look at the early nuclear events underlying pluripotent reprogramming. Here, we describe a polyethylene glycol (PEG)-mediated cell fusion protocol to generate interspecies heterokaryons and intraspecies hybrids between ES cells and B lymphocytes or fibroblasts. PMID:27659994

  11. Reprogramming of Somatic Cells Towards Pluripotency by Cell Fusion.

    PubMed

    Malinowski, Andrzej R; Fisher, Amanda G

    2016-01-01

    Pluripotent reprogramming can be dominantly induced in a somatic nucleus upon fusion with a pluripotent cell such as embryonic stem (ES) cell. Cell fusion between ES cells and somatic cells results in the formation of heterokaryons, in which the somatic nuclei begin to acquire features of the pluripotent partner. The generation of interspecies heterokaryons between mouse ES- and human somatic cells allows an experimenter to distinguish the nuclear events occurring specifically within the reprogrammed nucleus. Therefore, cell fusion provides a simple and rapid approach to look at the early nuclear events underlying pluripotent reprogramming. Here, we describe a polyethylene glycol (PEG)-mediated cell fusion protocol to generate interspecies heterokaryons and intraspecies hybrids between ES cells and B lymphocytes or fibroblasts.

  12. Endomembrane-associated RSD-3 is important for RNAi induced by extracellular silencing RNA in both somatic and germ cells of Caenorhabditis elegans

    PubMed Central

    Imae, Rieko; Dejima, Katsufumi; Kage-Nakadai, Eriko; Arai, Hiroyuki; Mitani, Shohei

    2016-01-01

    RNA silencing signals in C. elegans spread among cells, leading to RNAi throughout the body. During systemic spread of RNAi, membrane trafficking is thought to play important roles. Here, we show that RNAi Spreading Defective-3 (rsd-3), which encodes a homolog of epsinR, a conserved ENTH (epsin N-terminal homology) domain protein, generally participates in cellular uptake of silencing RNA. RSD-3 is previously thought to be involved in systemic RNAi only in germ cells, but we isolated several deletion alleles of rsd-3, and found that these mutants are defective in the spread of silencing RNA not only into germ cells but also into somatic cells. RSD-3 is ubiquitously expressed, and intracellularly localized to the trans-Golgi network (TGN) and endosomes. Tissue-specific rescue experiments indicate that RSD-3 is required for importing silencing RNA into cells rather than exporting from cells. Structure/function analysis showed that the ENTH domain alone is sufficient, and membrane association of the ENTH domain is required, for RSD-3 function in systemic RNAi. Our results suggest that endomembrane trafficking through the TGN and endosomes generally plays an important role in cellular uptake of silencing RNA. PMID:27306325

  13. Endomembrane-associated RSD-3 is important for RNAi induced by extracellular silencing RNA in both somatic and germ cells of Caenorhabditis elegans.

    PubMed

    Imae, Rieko; Dejima, Katsufumi; Kage-Nakadai, Eriko; Arai, Hiroyuki; Mitani, Shohei

    2016-06-16

    RNA silencing signals in C. elegans spread among cells, leading to RNAi throughout the body. During systemic spread of RNAi, membrane trafficking is thought to play important roles. Here, we show that RNAi Spreading Defective-3 (rsd-3), which encodes a homolog of epsinR, a conserved ENTH (epsin N-terminal homology) domain protein, generally participates in cellular uptake of silencing RNA. RSD-3 is previously thought to be involved in systemic RNAi only in germ cells, but we isolated several deletion alleles of rsd-3, and found that these mutants are defective in the spread of silencing RNA not only into germ cells but also into somatic cells. RSD-3 is ubiquitously expressed, and intracellularly localized to the trans-Golgi network (TGN) and endosomes. Tissue-specific rescue experiments indicate that RSD-3 is required for importing silencing RNA into cells rather than exporting from cells. Structure/function analysis showed that the ENTH domain alone is sufficient, and membrane association of the ENTH domain is required, for RSD-3 function in systemic RNAi. Our results suggest that endomembrane trafficking through the TGN and endosomes generally plays an important role in cellular uptake of silencing RNA.

  14. Endomembrane-associated RSD-3 is important for RNAi induced by extracellular silencing RNA in both somatic and germ cells of Caenorhabditis elegans.

    PubMed

    Imae, Rieko; Dejima, Katsufumi; Kage-Nakadai, Eriko; Arai, Hiroyuki; Mitani, Shohei

    2016-01-01

    RNA silencing signals in C. elegans spread among cells, leading to RNAi throughout the body. During systemic spread of RNAi, membrane trafficking is thought to play important roles. Here, we show that RNAi Spreading Defective-3 (rsd-3), which encodes a homolog of epsinR, a conserved ENTH (epsin N-terminal homology) domain protein, generally participates in cellular uptake of silencing RNA. RSD-3 is previously thought to be involved in systemic RNAi only in germ cells, but we isolated several deletion alleles of rsd-3, and found that these mutants are defective in the spread of silencing RNA not only into germ cells but also into somatic cells. RSD-3 is ubiquitously expressed, and intracellularly localized to the trans-Golgi network (TGN) and endosomes. Tissue-specific rescue experiments indicate that RSD-3 is required for importing silencing RNA into cells rather than exporting from cells. Structure/function analysis showed that the ENTH domain alone is sufficient, and membrane association of the ENTH domain is required, for RSD-3 function in systemic RNAi. Our results suggest that endomembrane trafficking through the TGN and endosomes generally plays an important role in cellular uptake of silencing RNA. PMID:27306325

  15. Ophiobolin A from Bipolaris oryzae perturbs motility and membrane integrities of porcine sperm and induces cell death on mammalian somatic cell lines.

    PubMed

    Bencsik, Ottó; Papp, Tamás; Berta, Máté; Zana, Annamária; Forgó, Péter; Dombi, György; Andersson, Maria A; Salkinoja-Salonen, Mirja; Vágvölgyi, Csaba; Szekeres, András

    2014-09-01

    Bipolaris oryzae is a phytopathogenic fungus causing a brown spot disease in rice, and produces substance that strongly perturbs motility and membrane integrities of boar spermatozoa. The substance was isolated from the liquid culture of the fungal strain using extraction and a multi-step semi-preparative HPLC procedures. Based on the results of mass spectrometric and 2D NMR techniques, the bioactive molecule was identified as ophiobolin A, a previously described sesterterpene-type compound. The purified ophiobolin A exhibited strong motility inhibition and viability reduction on boar spermatozoa. Furthermore, it damaged the sperm mitochondria significantly at sublethal concentration by the dissipation of transmembrane potential in the mitochondrial inner membrane, while the plasma membrane permeability barrier remained intact. The study demonstrated that the cytotoxicity of ophiobolin A toward somatic cell lines is higher by 1-2 orders of magnitude compared to other mitochondriotoxic mycotoxins, and towards sperm cells unique by replacing the progressive motility by shivering tail beating at low exposure concentration. PMID:25251540

  16. Somatic embryo-like structures of strawberry regenerated in vitro on media supplemented with 2,4-D and BAP.

    PubMed

    Omar, Genesia F; Mohamed, Fouad H; Haensch, Klaus-Thomas; Sarg, Sawsan H; Morsey, Mohamed M

    2013-09-01

    Somatic embryo-like structures (SELS) were produced in vitro from leaf disk and petiole explants of two cultivars of strawberry (Fragaria x ananassa Duch) on Murashige and Skoog medium with different concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and sucrose to check the embryonic nature of these structures histologically. A large number of SELS could be regenerated in both cultivars on media with 2-4 mg L(-1) 2,4-D in combination with 0.5 -1 mg L(-1) BAP and 50 g x L(-1) sucrose. Histological examination of SELS revealed the absence of a root pole. Therefore these structures cannot be strictly classified as somatic embryos. The SELS formed under the tested culture conditions represent malformed shoot-like and leaf-like structures. The importance of these results for the propagation of strawberries via somatic embryogenesis is discussed. PMID:24377134

  17. Somatic embryo-like structures of strawberry regenerated in vitro on media supplemented with 2,4-D and BAP.

    PubMed

    Omar, Genesia F; Mohamed, Fouad H; Haensch, Klaus-Thomas; Sarg, Sawsan H; Morsey, Mohamed M

    2013-09-01

    Somatic embryo-like structures (SELS) were produced in vitro from leaf disk and petiole explants of two cultivars of strawberry (Fragaria x ananassa Duch) on Murashige and Skoog medium with different concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and sucrose to check the embryonic nature of these structures histologically. A large number of SELS could be regenerated in both cultivars on media with 2-4 mg L(-1) 2,4-D in combination with 0.5 -1 mg L(-1) BAP and 50 g x L(-1) sucrose. Histological examination of SELS revealed the absence of a root pole. Therefore these structures cannot be strictly classified as somatic embryos. The SELS formed under the tested culture conditions represent malformed shoot-like and leaf-like structures. The importance of these results for the propagation of strawberries via somatic embryogenesis is discussed.

  18. Changes in the Essential Oil Components during the Development of Fennel Plants from Somatic Embryoids.

    PubMed

    Miura, Y; Ogawa, K; Fukui, H; Tabata, M

    1987-02-01

    Quantitative and qualitative changes of essential oils during the development of clonal plants of fennel propagated through somatic embryogenesis were investigated. Although no essential oil could be detected either in cultured cells or in somatic embryoids, monoter-penes such as alpha-phellandrene and alpha-pinene were found in radical leaves of regenerated plantlets cultured on a hormone-free agar medium. The regenerated plants cultivated in the field for about one month accumulated phenylpropanoids such as estragole, anethole, and fenchone in addition to the two monoterpenes described above in radical leaves. Rich accumulations of phenylpropanoids and monoterpenes were observed in the fruits; especially the contents of estragole and anethole were much higher than in radical leaves. PMID:17268973

  19. Suppressive effect of post- or pre-treatment of aspirin metabolite on mitomycin C-induced genotoxicity using the somatic mutation and recombination test in Drosophila melanogaster.

    PubMed

    Niikawa, Miki; Shin, Seizai; Nagase, Hisamitsu

    2007-01-01

    In our previous paper, we found that aspirin suppressed in a somatic mutation and recombination test (SMART) of mitomycin C (MMC) in Drosophila melanogaster. In order to reveal the mechanism of bio-antimutagenicity and/or preventive effect of aspirin, we evaluated the suppressive ability of each aspirin metabolite, such as salicylic acid (SA), salicyluric acid (SUA), gentisic acid (GA), gentisuric acid (GUA) and 2,3-dihydroxybenzoic acid (DHBA), in SMART in D. melanogaster using post- and pre-treatments. As for the post-treatment, SA reduced the numbers of large single and twin spots. GA reduced the small single and large single spots, and GUA reduced the single spots, large single and twin spots. The inhibition of GUA is slightly stronger than that of any other metabolites; the inhibition percentage is 49 at the dose of 5 mg/bottle. On the other hand, as for the pre-treatment, aspirin, SUA, GA and DHBA reduced the numbers of small single spots. SUA, GE and DHBA reduced the number of large single spots. Aspirin and its metabolites did not reduce the number of twin spots. The results of the present study suggest that SA, GA and GUA repair or replicate DNA-damage by MMC and SUA, GA, GUA and DHBA prevent DNA-damage by MMC. It is suggested that secondary cancer is prevented by aspirin post-treatment without losing the medicinal effectiveness (anti-tumor activity). Therefore, we consider there are effective doses and/or administration timing of aspirin and MMC to prevent secondary cancer. PMID:17275250

  20. Interpersonal psychotherapy for somatizing patients.

    PubMed

    Stuart, Scott; Noyes, Russell

    2006-01-01

    The interpersonal model is important for understanding somatizing behavior. According to this model, somatizing behavior is a form of interpersonal communication driven by insecure attachment. Interpersonal psychotherapy (IPT) is a time-limited, manual-based treatment designed to relieve symptoms and improve interpersonal functioning. Based on our experience using IPT with somatizing patients, we recommend a series of strategies for its successful implementation. These strategies include an emphasis on the therapeutic alliance within a bilaterally negotiated treatment contract, and aiming for improvement in interpersonal functioning rather than for alleviation of physical symptoms. Specific techniques include the use of bridging metaphors, communication analysis, and genuine positive reinforcement. With a focus on communication in a time-limited frame, fostered by a strong collaborative relationship, IPT appears to be a promising method of reducing somatizing behavior. We urge further research on the efficacy of this form of therapy. PMID:16785770

  1. Differences in protodermal cell wall structure in zygotic and somatic embryos of Daucus carota (L.) cultured on solid and in liquid media.

    PubMed

    Dobrowolska, Izabela; Majchrzak, Oliwia; Baldwin, Timothy C; Kurczynska, Ewa U

    2012-01-01

    The ultrastructure, cuticle, and distribution of pectic epitopes in outer periclinal walls of protodermal cells of Daucus carota zygotic and somatic embryos from solid and suspension culture were investigated. Lipid substances were present as a continuous layer in zygotic and somatic embryos cultured on solid medium. Somatic embryos from suspension cultures were devoid of cuticle. The ultrastructure of the outer walls of protodermis of embryos was similar in zygotic and somatic embryos from solid culture. Fibrillar material was observed on the surface of somatic embryos. In zygotic embryos, in cotyledons and root pectic epitopes recognised by the antibody JIM5 were observed in all cell walls. In hypocotyls of these embryos, these pectic epitopes were not present in the outer periclinal and anticlinal walls of the protodermis. In somatic embryos from solid media, distribution of pectic epitopes recognised by JIM5 was similar to that described for their zygotic counterparts. In somatic embryos from suspension culture, pectic epitopes recognised by JIM5 were detected in all cell walls. In the cotyledons and hypocotyls, a punctate signal was observed on the outside of the protodermis. Pectic epitopes recognised by JIM7 were present in all cell walls independent of embryo organs. In zygotic embryos, this signal was punctate; in somatic embryos from both cultures, this signal was uniformly distributed. In embryos from suspension cultures, a punctate signal was detected outside the surface of cotyledon and hypocotyl. These data are discussed in light of current models for embryogenesis and the influence of culture conditions on cell wall structure.

  2. 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

  3. Somatic cell mutagenesis in Drosophila: recovery of genetic damage in relation to the types of DNA lesions induced in mutationally unstable and stable X-chromosomes.

    PubMed

    Vogel, E W

    1989-03-01

    This paper reports the results of a study on the genotoxic activities of 12 mutagens and clastogens of widely differing mode of action in somatic cells in vivo, i.e., in the eye primordia of Drosophila larvae. After emergence, adult flies were monitored for aberrantly colored sectors in the compound eyes of the following genotypes: UZ males and females (zeste) carrying a genetically unstable transposable element, SZ males and females (zeste) carrying a partial duplication of the w+ locus plus a transposon insert, white-coral/white (wco/w) females, w+/w females and w+ males. The UZ and SZ marker sets make it possible to monitor shifts from zeste to red (scored as mosaic red spots, RS) and for loss of the white locus (light spots, LS). wco/w+ females were scored for mosaic twin spots (TS) and LS, w+ genotypes for just LS. The genotoxins analyzed were methyl methanesulfonate (MMS), dimethyl sulfate (DMS) and ethylnitrosourea (ENU) (alkylating), adriamycin (AM) and daunomycin (DM) (intercalating), Trenimon, Thio-TEPA and cisplatin (DDP) (cross-linking), bleomycin (strand-breaking), 7,12-dimethylbenz[a]anthracene (DMBA) and 9,10-dimethylanthracene (DA) (bulky monoadducts) and cytosine arabinofuranoside (inhibition of DNA synthesis). The relative mutabilities with frequencies of mosaic light spots (LS) in w+/w female as the standard (relative mutability = 1) vs. genotypes UZ (RS in male) vs. SZ (RS in male) vs. w+ (LS in male) were 1:0.6:0.2:0.3 for MMS, 1:0.09:0.05:0.7 for DDP, and 1:1.6:0.2:1.0 for ENU, ENU showed exceptional behavior in that it was the only compound for which mutational response, measured by the induction of red spots, was highest with the UZ marker set. Occurrence of large light spots (LS) in male but not in female genotypes was negatively correlated with efficiency of agents for chromosomal damage, suggesting that in the hemizygous condition, as in males, selection of damaged cells and mitotic delay may have played a significant role. In general

  4. 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

  5. Embryogenesis and plant regeneration of pakchoi (Brassica rapa L. ssp. chinensis) via in vitro isolated microspore culture.

    PubMed

    Cao, M Q; Li, Y; Liu, F; Doré, C

    1994-05-01

    Isolated microspores of various populations of three varieties of the Chinese cabbage pakchoi (Brassica rapa ssp. chinensis) were cultivated in vitro on NLN82 medium (Lichter 1982) and embryos and plantlets obtained with nine cultivars. The best embryo yield per bud was 57.4. A 33°C one day heat treatment was generally necessary to induce embryogenesis. Analysis of ploidy level through flow cytometry for two cultivars indicated that haploids were present.

  6. 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

  7. 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.

  8. 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

  9. Cryopreservation of zygotic embryo axes and somatic embryos of European chestnut.

    PubMed

    Corredoira, Elena; San-José, M Carmen; Ballester, Antonio; Vieitez, Ana M

    2004-01-01

    This work describes experiments demonstrating the feasibility of long-term conservation of Castanea sativa germplasm through cryopreservation of embryonic axes or somatic embryo clumps. Between 93 % and 100 % of excised embryonic axes of recalcitrant chestnut seeds survived storage in liquid nitrogen (LN) following desiccation in a laminar flow cabinet to moisture contents of 20-24 % (on a fresh weight basis), and some 63 % subsequently developed as whole plants. Desiccation to moisture contents less than 19 % produced damage resulting in loss of organized plant development after cryostorage, allowing only root growth. When 6-8 mg clumps of globular or heart-shaped somatic embryos were precultured for 7 days on high-sucrose medium and then desiccated to a moisture content of 25 % before storage in LN, the embryogenesis resumption level after thawing was 33 %. When the embryo clumps were precultured for 3 days on high-sucrose medium followed by 60 min application of PVS2 vitrification solution before cryostorage, the post-storage embryogenesis resumption level was 68 %.

  10. 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.

  11. Physiological inputs regulate species-specific anatomy during embryogenesis and regeneration

    PubMed Central

    Sullivan, Kelly G.; Emmons-Bell, Maya; Levin, Michael

    2016-01-01

    ABSTRACT A key problem in evolutionary developmental biology is identifying the sources of instructive information that determine species-specific anatomical pattern. Understanding the inputs to large-scale morphology is also crucial for efforts to manipulate pattern formation in regenerative medicine and synthetic bioengineering. Recent studies have revealed a physiological system of communication among cells that regulates pattern during embryogenesis and regeneration in vertebrate and invertebrate models. Somatic tissues form networks using the same ion channels, electrical synapses, and neurotransmitter mechanisms exploited by the brain for information-processing. Experimental manipulation of these circuits was recently shown to override genome default patterning outcomes, resulting in head shapes resembling those of other species in planaria and Xenopus. The ability to drastically alter macroscopic anatomy to that of other extant species, despite a wild-type genomic sequence, suggests exciting new approaches to the understanding and control of patterning. Here, we review these results and discuss hypotheses regarding non-genomic systems of instructive information that determine biological growth and form. PMID:27574538

  12. Physiological inputs regulate species-specific anatomy during embryogenesis and regeneration.

    PubMed

    Sullivan, Kelly G; Emmons-Bell, Maya; Levin, Michael

    2016-01-01

    A key problem in evolutionary developmental biology is identifying the sources of instructive information that determine species-specific anatomical pattern. Understanding the inputs to large-scale morphology is also crucial for efforts to manipulate pattern formation in regenerative medicine and synthetic bioengineering. Recent studies have revealed a physiological system of communication among cells that regulates pattern during embryogenesis and regeneration in vertebrate and invertebrate models. Somatic tissues form networks using the same ion channels, electrical synapses, and neurotransmitter mechanisms exploited by the brain for information-processing. Experimental manipulation of these circuits was recently shown to override genome default patterning outcomes, resulting in head shapes resembling those of other species in planaria and Xenopus. The ability to drastically alter macroscopic anatomy to that of other extant species, despite a wild-type genomic sequence, suggests exciting new approaches to the understanding and control of patterning. Here, we review these results and discuss hypotheses regarding non-genomic systems of instructive information that determine biological growth and form. PMID:27574538

  13. 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

  14. 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

  15. Embryogenesis induction, callogenesis, and plant regeneration by in vitro culture of tomato isolated microspores and whole anthers.

    PubMed

    Seguí-Simarro, José M; Nuez, Fernando

    2007-01-01

    In this work, some of the different in vitro developmental pathways into which tomato microspores or microsporocytes can be deviated experimentally were explored. The two principal ones are direct embryogenesis from isolated microspores and callus formation from meiocyte-containing anthers. By means of light and electron microscopy, the process of early embryogenesis from isolated microspores and the disruption of normal meiotic development and change of developmental fate towards callus proliferation, morphogenesis, and plant regeneration have been shown. From microspores isolated at the vacuolate stage, embryos can be directly induced, thus avoiding non-androgenic products. In contrast, several different morphogenic events can be triggered in cultures of microsporocyte-containing anthers under adequate conditions, including indirect embryogenesis, adventitious organogenesis, and plant regeneration. Both callus and regenerated plants may be haploid, diploid, and mostly mixoploid. The results demonstrate that both gametophytic and sporophytic calli occur in cultured tomato anthers, and point to an in vitro-induced disturbance of cytokinesis and subsequent fusion of daughter nuclei as a putative cause for mixoploidy and genome doubling during both tetrad compartmentalization and callus proliferation. The potential implications of the different alternative pathways are discussed in the context of their application to the production of doubled-haploid plants in tomato, which is still very poorly developed.

  16. Somatic Symptom and Related Disorders

    MedlinePlus

    ... and symptoms a person feels are related to psychological factors. These symptoms can't be traced to a specific physical cause. In people who have a somatic symptom and related disorder, medical test results are either normal or don't explain ...

  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. Somatic and dendritic perforated-patch recordings reveal b-adrenergic receptor-induced depolarization in medial prefrontal cortex pyramidal neurons.

    PubMed

    Szulczyk, Bartłomiej

    2016-01-01

    The aim of this perforated-patch study was to test the effect of isoproterenol on the membrane potential in mPFC (medial prefrontal cortex) pyramidal neurons. Isoproterenol depolarized the membrane potential recorded from the soma. This effect was absent in the presence of metoprolol, suggesting the involvement of beta1-adrenergic receptors. The adenylate cyclase activator forskolin also depolarized the membrane potential. Moreover, the effect of isoproterenol was abolished by the adenylate cyclase inhibitor SQ 22536. This suggested that adenylate cyclase was involved in mediating the effect of the beta-adrenergic receptor agonist. The isoproterenol-induced depolarization persisted after inhibition of protein kinase A with H-89. The effect of beta-adrenergic receptor activation on the membrane potential was dependent on Ih channels because it was abolished in the presence of the Ih channel inhibitor ZD 7288. Dendritic recordings were also performed. In the dendritic segments between 100 microm and 150 microm from the soma and between 200 microm and 250 microm from the soma, isoproterenol also depolarized the membrane potential. The magnitude of the beta-adrenergic receptor-stimulated depolarization was the same in the soma and in both dendritic localizations. The depolarization exerted by isoproterenol may influence PFC cognitive functions. PMID:27373953

  19. Microspore Embryogenesis Through Anther Culture in Citrus clementina Hort. ex Tan.

    PubMed

    Chiancone, Benedetta; Germanà, Maria Antonietta

    2016-01-01

    Anther culture is a biotechnological method that allows to obtain, in one step, homozygous plants, very important to plant breeding, due to their numerous applications in mutation research, selection, genome sequencing, genetic analysis, and transformation. To induce the microspores, i.e., the immature male gametes, to switch from the normal gametophytic pathway to the sporophytic one, it is necessary to submit them to a type of stress, such as high or low temperature, starvation, or magnetic field. Stress can be applied to the donor plants and/or the floral buds or the anthers or the isolated microspores, before or during the culture. In this chapter, the protocol to induce gametic embryogenesis from anther culture of several cultivars of Citrus clementina Hort. ex Tan. is reported.

  20. Microspore Embryogenesis Through Anther Culture in Citrus clementina Hort. ex Tan.

    PubMed

    Chiancone, Benedetta; Germanà, Maria Antonietta

    2016-01-01

    Anther culture is a biotechnological method that allows to obtain, in one step, homozygous plants, very important to plant breeding, due to their numerous applications in mutation research, selection, genome sequencing, genetic analysis, and transformation. To induce the microspores, i.e., the immature male gametes, to switch from the normal gametophytic pathway to the sporophytic one, it is necessary to submit them to a type of stress, such as high or low temperature, starvation, or magnetic field. Stress can be applied to the donor plants and/or the floral buds or the anthers or the isolated microspores, before or during the culture. In this chapter, the protocol to induce gametic embryogenesis from anther culture of several cultivars of Citrus clementina Hort. ex Tan. is reported. PMID:26619882

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. The histone chaperone CAF-1 safeguards somatic cell identity

    PubMed Central

    Cheloufi, Sihem; Elling, Ulrich; Hopfgartner, Barbara; Jung, Youngsook L; Murn, Jernej; Ninova, Maria; Hubmann, Maria; Badeaux, Aimee I; Ang, Cheen Euong; Tenen, Danielle; Wesche, Daniel J; Abazova, Nadezhda; Hogue, Max; Tasdemir, Nilgun; Brumbaugh, Justin; Rathert, Philipp; Jude, Julian; Ferrari, Francesco; Blanco, Andres; Fellner, Michaela; Wenzel, Daniel; Zinner, Marietta; Vidal, Simon E; Bell, Oliver; Stadtfeld, Matthias; Chang, Howard Y.; Almouzni, Genevieve; Lowe, Scott W; Rinn, John; Wernig, Marius; Aravin, Alexei; Shi, Yang; Park, Peter; Penninger, Josef M; Zuber, Johannes; Hochedlinger, Konrad

    2016-01-01

    Cellular differentiation involves profound remodeling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNAi screens targeting chromatin factors during transcription factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPSCs). Remarkably, subunits of the chromatin assembly factor-1 (CAF-1) complex emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPSC formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 as a novel regulator of somatic cell identity during transcription factor-induced cell fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting. PMID:26659182

  6. Photodynamic inactivation of somatic frog nerve ex vivo

    NASA Astrophysics Data System (ADS)

    Akchurin, Garif G.; Seliverstov, George A.; Akchurin, George G.; Kudryashova, Svetlana Y.

    2004-06-01

    New techniques research mechanisms of photdynamic reactions at somatic frog nerve was approved. Dosimetry PDT with minimum time resolution ~1ms determined by changing the amplitude of compound action potential of somatic frog nerve. Light-induced inactivation of dynamic response of somatic frog nerve on electrical pulsed excitation was study ex vivo. The light-sensitive dyes: methylene blue (Mb), Indocianin green and eryhtrocin-B has been used on photodynamic induced inactivation of the processes generation nerve pulses. Inactivation of consequence action potential of somatic frog nerve using excitation of electical pulsed was achieved by irradiation with He-Ne laser light in a red spectral region (λ=633 nm, power level 2-20 mW), diode laser (λ=805 nm, P<0.1-1 W/cm2) in the case of Indocianin green and YAG:Nd laser (λ=532 nm, P~1mW) for eryhtrocin-B. It was discovered that methylene blue, Indocainine green and erytrocin-B decrease of the amplitude compound action potential of the ensemble neurons. The possible cell death mechanism was connected with damage of the sodium potassium adenosine triphosphatase (K-Na ATP) active transport which decrease of amplitude of compound action potential and decrease lifetime ionic channel of membrane nerve.

  7. 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.

  8. Cryopreservation of zygotic embryonic axes and somatic embryos of European chestnut.

    PubMed

    Vieitez, Ana M; San-José, M Carmen; Corredoira, Elena

    2011-01-01

    For Castanea sativa (European chestnut), a species with recalcitrant seeds that is not easily propagated vegetatively, cryopreservation is one of the most promising techniques for maintaining genetic resource diversity and for conservation of selected germplasms. Long-term conservation of selected seeds and valuable embryogenic lines can be achieved through the cryopreservation of zygotic embryonic axes and somatic embryos, respectively. This chapter describes methods for the desiccation-based cryostorage of zygotic embryonic axes, and the vitrification-based cryopreservation of somatic embryos. For zygotic embryonic axes, the highest post-thaw survival and plantlet recovery rates are obtained by desiccation in a laminar flow hood to 20-25% moisture content, followed by direct immersion in liquid nitrogen. For somatic embryos, embryogenesis resumption rates of over 60% are achieved by preculture of embryo clumps for 3 days on solid medium containing 0.3 M sucrose, incubation in PVS2 vitrification solution for 60 min at 0°C, and direct immersion in liquid nitrogen. Plantlet recovery from cryostored embryogenic lines requires proliferation of the thawed embryos and subsequent maturation before germination and conversion into plantlets.

  9. Histone variant macroH2A marks embryonic differentiation in vivo and acts as an epigenetic barrier to induced pluripotency

    PubMed Central

    Pasque, Vincent; Radzisheuskaya, Aliaksandra; Gillich, Astrid; Halley-Stott, Richard P.; Panamarova, Maryna; Zernicka-Goetz, Magdalena; Surani, M. Azim; Silva, José C. R.

    2012-01-01

    Summary How cell fate becomes restricted during somatic cell differentiation is a long-lasting question in biology. Epigenetic mechanisms not present in pluripotent cells and acquired during embryonic development are expected to stabilize the differentiated state of somatic cells and thereby restrict their ability to convert to another fate. The histone variant macroH2A acts as a component of an epigenetic multilayer that heritably maintains the silent X chromosome and has been shown to restrict tumor development. Here we show that macroH2A marks the differentiated cell state during mouse embryogenesis. MacroH2A.1 was found to be present at low levels upon the establishment of pluripotency in the inner cell mass and epiblast, but it was highly enriched in the trophectoderm and differentiated somatic cells later in mouse development. Chromatin immunoprecipitation revealed that macroH2A.1 is incorporated in the chromatin of regulatory regions of pluripotency genes in somatic cells such as mouse embryonic fibroblasts and adult neural stem cells, but not in embryonic stem cells. Removal of macroH2A.1, macroH2A.2 or both increased the efficiency of induced pluripotency up to 25-fold. The obtained induced pluripotent stem cells reactivated pluripotency genes, silenced retroviral transgenes and contributed to chimeras. In addition, overexpression of macroH2A isoforms prevented efficient reprogramming of epiblast stem cells to naïve pluripotency. In summary, our study identifies for the first time a link between an epigenetic mark and cell fate restriction during somatic cell differentiation, which helps to maintain cell identity and antagonizes induction of a pluripotent stem cell state. PMID:23077180

  10. Cryopreservation of plumular explants of coconut (Cocos nucifera L.) to support programmes for mass clonal propagation through somatic embryogenesis.

    PubMed

    Hornung, R; Domas, R; Lynch, P T

    2001-01-01

    Callus growth has been observed from plumules of ecotype Laguna Tall after cryopreservation using an encapsulation/dehydration protocol. Sucrose preculture treatment and silica gel dehydration both significantly influenced the frequency of callus formation from non-frozen and frozen plumules. The greatest frequency of post-thaw callus growth occurred after incubation of the encapsulated plumules for 72-96 h in medium containing 0.75 M sucrose followed by desiccation over silica gel for 7-8 h down to approximately 30% moisture content (fresh weight basis). Freezing and thawing were carried out rapidly. Post-thaw recovery rates in excess of 80% were recorded.

  11. Characterization of two novel small molecules targeting melanocyte development in zebrafish embryogenesis.

    PubMed

    Chen, Lu; Ren, Xi; Liang, Fang; Li, Song; Zhong, Hanbing; Lin, Shuo

    2012-07-01

    Melanocytes are pigment cells that are closely associated with many skin disorders, such as vitiligo, piebaldism, Waardenburg syndrome, and the deadliest skin cancer, melanoma. Through studies of model organisms, the genetic regulatory network of melanocyte development during embryogenesis has been well established. This network also seems to be shared with adult melanocyte regeneration and melanoma formation. To identify chemical regulators of melanocyte development and homeostasis, we screened a small-molecule library of 6000 compounds using zebrafish embryos and identified five novel compounds that inhibited pigmentation. Here we report characterization of two compounds, 12G9 and 36E9, which disrupted melanocyte development. TUNEL assay indicated that these two compounds induced apoptosis of melanocytes. Furthermore, compound 12G9 specifically inhibited the viability of mammalian melanoma cells in vitro. These two compounds should be useful as chemical biology tools to study melanocytes and could serve as drug candidates against melanocyte-related diseases.

  12. In vivo imaging of zebrafish embryogenesis

    PubMed Central

    Keller, Philipp J.

    2013-01-01

    The zebrafish Danio rerio has emerged as a powerful vertebrate model system that lends itself particularly well to quantitative investigations with live imaging approaches, owing to its exceptionally high optical clarity in embryonic and larval stages. Recent advances in light microscopy technology enable comprehensive analyses of cellular dynamics during zebrafish embryonic development, systematic mapping of gene expression dynamics, quantitative reconstruction of mutant phenotypes and the system-level biophysical study of morphogenesis. Despite these technical breakthroughs, it remains challenging to design and implement experiments for in vivo long-term imaging at high spatio-temporal resolution. This article discusses the fundamental challenges in zebrafish long-term live imaging, provides experimental protocols and highlights key prop1erties and capabilities of advanced fluorescence microscopes. The article focuses in particular on experimental assays based on light sheet-based fluorescence microscopy, an emerging imaging technology that achieves exceptionally high imaging speeds and excellent signal-to-noise ratios, while minimizing light-induced damage to the specimen. This unique combination of capabilities makes light sheet microscopy an indispensable tool for the in vivo long-term imaging of large developing organisms. PMID:23523701

  13. 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

  14. Mcm10 is required for oogenesis and early embryogenesis in Drosophila.

    PubMed

    Reubens, Michael C; Biller, Megan D; Bedsole, Sidney E; Hopkins, Lucas T; Ables, Elizabeth T; Christensen, Tim W

    2015-11-01

    Efficient replication of the genome and the establishment of endogenous chromatin states are processes that are essential to eukaryotic life. It is well documented that Mcm10 is intimately linked to both of these important biological processes; therefore, it is not surprising that Mcm10 is commonly misregulated in many human cancers. Most of the research regarding the biological roles of Mcm10 has been performed in single-cell or cell-free in-vitro systems. Though these systems are informative, they are unable to provide information on the cell-specific function of Mcm10 in the context of the tissue and organ systems that comprise multicellular eukaryotes. We therefore sought to identify the potential biological functions of Mcm10 in the context of a complex multicellular organism by continuing our analysis in Drosophila using three novel hypomorphic alleles. Observation of embryonic nuclear morphology and quantification of embryo hatch rates reveal that maternal loading of Mcm10 is required for embryonic nuclear stability, and suggest a role for Mcm10 post zygotic transition. Contrary to the essential nature of Mcm10 depicted in the literature, it does not appear to be required for adult viability in Drosophila if embryonic requirements are met. Although not required for adult somatic viability, analysis of fecundity and ovarian morphology in mutant females suggest that Mcm10 plays a role in maintenance of the female germline. Taken together, our results demonstrate critical roles for Mcm10 during early embryogenesis, and mark the first data linking Mcm10 to female specific reproduction in multicellular eukaryotes. PMID:26369283

  15. 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.

  16. Genome-wide analysis reveals divergent patterns of gene expression during zygotic and somatic embryo maturation of Theobroma cacao L., the chocolate tree

    PubMed Central

    2014-01-01

    Background Theobroma cacao L. is a tropical fruit tree, the seeds of which are used to create chocolate. In vitro somatic embryogenesis (SE) of cacao is a propagation system useful for rapid mass-multiplication to accelerate breeding programs and to provide plants directly to farmers. Two major limitations of cacao SE remain: the efficiency of embryo production is highly genotype dependent and the lack of full cotyledon development results in low embryo to plant conversion rates. With the goal to better understand SE development and to improve the efficiency of SE conversion we examined gene expression differences between zygotic and somatic embryos using a whole genome microarray. Results The expression of 28,752 genes was determined at 4 developmental time points during zygotic embryogenesis (ZE) and 2 time points during cacao somatic embryogenesis (SE). Within the ZE time course, 10,288 differentially expressed genes were enriched for functions related to responses to abiotic and biotic stimulus, metabolic and cellular processes. A comparison ZE and SE expression profiles identified 10,175 differentially expressed genes. Many TF genes, putatively involved in ethylene metabolism and response, were more strongly expressed in SEs as compared to ZEs. Expression levels of genes involved in fatty acid metabolism, flavonoid biosynthesis and seed storage protein genes were also differentially expressed in the two types of embryos. Conclusions Large numbers of genes were differentially regulated during various stages of both ZE and SE development in cacao. The relatively higher expression of ethylene and flavonoid related genes during SE suggests that the developing tissues may be experiencing high levels of stress during SE maturation caused by the in vitro environment. The expression of genes involved in the synthesis of auxin, polyunsaturated fatty acids and secondary metabolites was higher in SEs relative to ZEs despite lack of lipid and metabolite accumulation

  17. 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.

  18. Transposable DNA elements and life history traits: II. Transposition of P DNA elements in somatic cells reduces fitness, mating activity, and locomotion of Drosophila melanogaster.

    PubMed

    Woodruff, R C; Thompson, J N; Barker, J S; Huai, H

    1999-01-01

    Some transposable DNA elements in higher organisms are active in somatic cells, as well as in germinal cells. What effect does the movement of DNA elements in somatic cells have on life history traits? It has previously been reported that somatically active P and mariner elements in Drosophila induce genetic damage and significantly reduce lifespan. In this study, we report that the movement of P elements in somatic cells also significantly reduces fitness, mating activity, and locomotion of Drosophila melanogaster. If other elements cause similar changes in life history traits, it is doubtful if transposable DNA elements remain active for long in somatic cells in natural populations.

  19. Somatic complaints in childhood tic disorders.

    PubMed

    Frank, M S; Sieg, K G; Gaffney, G R

    1991-01-01

    Twenty-six children diagnosed with chronic tic disorders (18 with Gilles de la Tourette syndrome and 8 with chronic motor tic disorder) were studied for unexplained physical complaints. Compared to normal controls, an excess of somatic complaints was found in the tic disorders group; this was similar to an excess of somatic complaints in a mixed psychiatric clinic group. Medication produced no significant effect on somatic complaints for patients in the tic and psychiatric clinic groups. Within the tic disorders group, no significant correlation was found between the increased somatic complaints and the severity of anxiety, dysphoria, or movement disorder. PMID:1961851

  20. 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.

  1. Promotion of oogenesis and embryogenesis in the C. elegans gonad by EFL-1/DPL-1 (E2F) does not require LIN-35 (pRB).

    PubMed

    Chi, Woo; Reinke, Valerie

    2006-08-01

    In Caenorhabditis elegans, EFL-1 (E2F), DPL-1 (DP) and LIN-35 (pRb) act coordinately in somatic tissues to inhibit ectopic cell division, probably by repressing the expression of target genes. EFL-1, DPL-1 and LIN-35 are also present in the germline, but do not always act together. Strong loss-of-function mutations in either efl-1 or dpl-1 cause defects in oogenesis that result in sterility, while lin-35 mutants are fertile with reduced broods. Microarray-based expression profiling of dissected gonads from efl-1, dpl-1 and lin-35 mutants reveals that EFL-1 and DPL-1 promote expression of an extensively overlapping set of target genes, consistent with the expectation that these two proteins function as a heterodimer. Regulatory regions upstream of many of these target genes have a canonical E2F-binding site, suggesting that their regulation by EFL-1/DPL-1 is direct. Many EFL-1/DPL-1 responsive genes encode proteins required for oogenesis and early embryogenesis, rather than cell cycle components. By contrast, LIN-35 appears to function primarily as a repressor of gene expression in the germline, and the genes that it acts on are for the most part distinct from those regulated by EFL-1 and/or DPL-1. Thus, in vivo, C. elegans E2F directly promotes oogenesis and embryogenesis through the activation of a tissue-specific transcriptional program that does not require LIN-35.

  2. Arsenic Exposure to Killifish During Embryogenesis Alters Muscle Development

    PubMed Central

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

    2012-01-01

    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

  3. Reporter genes for embryogenesis research in livestock species.

    PubMed

    Habermann, F A; Wuensch, A; Sinowatz, F; Wolf, E

    2007-09-01

    Currently, our knowledge of early mammalian embryogenesis, stem cell differentiation and development is largely based on studies performed in mouse models. However, in important aspects, e.g. the timing of epigenetic reprogramming and embryonic genome activation, livestock species probably reflect far more closely the situation in men and other non-rodent mammals. A major challenge is the fact that in mammals, the development of individual zygotes is highly variable and vulnerable, and the outcome is uncertain. Valid indicators of the highly heterogeneous development and health status, and the actual developmental potential of individual oocytes, zygotes or embryos would be crucially important to tap the full power of holistic transcriptome and proteome analyses. Fluorescent reporter proteins opened new vistas for embryology and stem cell research: they can be used as reporters for the activity of gene promoters or tagged to functional proteins to study their intracellular localization in living cells, tissues and organisms. Fluorescent reporter genes may be used to microscopically observe key processes of early development. Thus, novel information related to developmental potential can be obtained from living embryos before processing them, e.g. for "-omic" studies. This review summarizes the main current reporter gene techniques and gene transfer approaches, which might be suitable for the investigation of early embryogenesis in livestock mammals. The potential of promoter reporter genes is exemplified by a bovine model system for quantitative monitoring of transcriptional reactivation of the so-called pluripotency gene POU5F1 in cloned bovine embryos.

  4. Cell morphodynamics visualization from images of zebrafish embryogenesis.

    PubMed

    Campana, Matteo; Sarti, Alessandro

    2010-07-01

    Laser scanning microscopy provides high-resolution nondestructive in vivo imaging to capture specific structures that have been fluorescently labeled, such as cellular nuclei and membranes, throughout early zebrafish embryogenesis. An increasingly challenging problem biologists must face is how to effectively explore, follow, and study the thousands of cells contained in the resulting time-varying volume data that are large in space, time, and variable domain. Visual data explorations, such as direct volume rendering, have been successfully used for the analysis of volumetric data. However, visualizing large-scale time-varying fields remains a challenging problem. In this paper we present a novel Focus+Context animated volume rendering. The technique is based on the distance map of objects of interest and on a scene graph architecture. We demonstrate that distance map driven volume rendering, implemented in modern graphics hardware, is suited to generate run time and interactive representations such as ghosted rendering and cut-away rendering. The experimental results on zebrafish embryogenesis data demonstrate that the technique is suited to uncover and to analyze biological events, such as organogenesis, contained in time-varying volumetric dataset.

  5. Plant regeneration of Korean wild ginseng (Panax ginseng Meyer) mutant lines induced by γ-irradiation ((60)Co) of adventitious roots.

    PubMed

    Zhang, Jun-Ying; Sun, Hyeon-Jin; Song, In-Ja; Bae, Tae-Woong; Kang, Hong-Gyu; Ko, Suk-Min; Kwon, Yong-Ik; Kim, Il-Woung; Lee, Jaechun; Park, Shin-Young; Lim, Pyung-Ok; Kim, Yong Hwan; Lee, Hyo-Yeon

    2014-07-01

    An efficient in vitro protocol has been established for somatic embryogenesis and plantlet conversion of Korean wild ginseng (Panax ginseng Meyer). Wild-type and mutant adventitious roots derived from the ginseng produced calluses on Murashige and Skoog (MS) medium supplemented with 0.5 mg/L 2,4-dichlorophenoxyacetic acid and 0.3 mg/L kinetin; 53.3% of the explants formed callus. Embryogenic callus proliferation and somatic embryo induction occurred on MS medium containing 0.5 mg/L 2,4-dichlorophenoxyacetic acid. The induced somatic embryos further developed to maturity on MS medium with 5 mg/L gibberellic acid, and 85% of them germinated. The germinated embryos were developed to shoots and elongated on MS medium with 5 mg/L gibberellic acid. The shoots developed into plants with well-developed taproots on one-third strength Schenk and Hildebrandt basal medium supplemented with 0.25 mg/L 1-naphthaleneacetic acid. When the plants were transferred to soil, about 30% of the regenerated plants developed into normal plants.

  6. 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…

  7. Muscle formation during embryogenesis of the polychaete Ophryotrocha diadema (Dorvilleidae) – new insights into annelid muscle patterns

    PubMed Central

    Bergter, Annette; Brubacher, John L; Paululat, Achim

    2008-01-01

    Background The standard textbook information that annelid musculature consists of oligochaete-like outer circular and inner longitudinal muscle-layers has recently been called into question by observations of a variety of complex muscle systems in numerous polychaete taxa. To clarify the ancestral muscle arrangement in this taxon, we compared myogenetic patterns during embryogenesis of Ophryotrocha diadema with available data on oligochaete and polychaete myogenesis. This work addresses the conflicting views on the ground pattern of annelids, and adds to our knowledge of the evolution of lophotrochozoan taxa. Results Somatic musculature in Ophryotrocha diadema can be classified into the trunk, prostomial/peristomial, and parapodial muscle complexes. The trunk muscles comprise strong bilateral pairs of distinct dorsal and ventral longitudinal strands. The latter are the first to differentiate during myogenesis. They originate within the peristomium and grow posteriorly through the continuous addition of myocytes. Later, the longitudinal muscles also expand anteriorly and form a complex arrangement of prostomial muscles. Four embryonic parapodia differentiate in an anterior-to-posterior progression, significantly contributing to the somatic musculature. Several diagonal and transverse muscles are present dorsally. Some of the latter are situated external to the longitudinal muscles, which implies they are homologous to the circular muscles of oligochaetes. These circular fibers are only weakly developed, and do not appear to form complete muscle circles. Conclusion Comparison of embryonic muscle patterns showed distinct similarities between myogenetic processes in Ophryotrocha diadema and those of oligochaete species, which allows us to relate the diverse adult muscle arrangements of these annelid taxa to each other. These findings provide significant clues for the interpretation of evolutionary changes in annelid musculature. PMID:18171469

  8. The molecular basis of somatic hypermutation of immunoglobulin genes.

    PubMed

    Storb, U

    1996-04-01

    Somatic hypermutation amplifies the variable region repertoire of immunoglobulin genes. Recent experimental evidence has thrown light on various molecular models of somatic hypermutation. A link between somatic hypermutation and transcription coupled DNA repair is shaping up.

  9. Somatic mosaicism in the human genome.

    PubMed

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

    2014-12-11

    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.

  10. 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

  11. TALEN mediated somatic mutagenesis in murine models of cancer

    PubMed Central

    Zhang, Shuyuan; Li, Lin; Kendrick, Sara L.; Gerard, Robert D.; Zhu, Hao

    2014-01-01

    Cancer genome sequencing has identified numerous somatic mutations whose biological relevance is uncertain. In this study, we used genome-editing tools to create and analyze targeted somatic mutations in murine models of liver cancer. TALEN were designed against β-catenin (Ctnnb1) and Apc, two commonly mutated genes in hepatocellular carcinoma (HCC), to generate isogenic HCC cell lines. Both mutant cell lines exhibited evidence of Wnt pathway dysregulation. We asked if these TALENs could create targeted somatic mutations after hydrodynamic transfection (HDT) into mouse liver. TALENs targeting β-catenin promoted endogenous HCC carrying the intended gain-of-function mutations. However, TALENs targeting Apc were not as efficient in inducing in vivo homozygous loss-of-function mutations. We hypothesized that hepatocyte polyploidy might be protective against TALEN-induced loss of heterozygosity (LOH), and indeed Apc gene editing was less efficient in tetraploid than in diploid hepatocytes. To increase efficiency, we administered adenoviral Apc TALENs and found that we could achieve a higher mutagenesis rate in vivo. Our results demonstrate that genome-editing tools can enable the in vivo study of cancer genes and faithfully recapitulate the mosaic nature of mutagenesis in mouse cancer models. PMID:25070752

  12. Quantitative and ultrastructural analysis of the chondriome in ovogenesis and embryogenesis of the sea urchin Paracentrotus lividus. 2. Growth and proliferation of mitochondria in embryogenesis.

    PubMed

    Sukhomlinova MYu; Kireyev, I I; Fais, D; Giudice, G; Polyakov VYu

    2001-07-01

    The dynamics of structural changes of the chondriome in the early development of the sea urchin Paracentrotus lividus was studied. Mature eggs and embryos at various stages of cleavage were used for quantitative and ultrastructural analysis based on computerized 3D reconstruction from serial ultrathin sections. The following structural transformations of the chondriome were shown to occur in the course of embryogenesis: (i) 15 min after fertilization, mitochondrial clusters disintegrate, and mitochondrial division is induced. At the stage of two blastomeres the population of mitochondria increases twofold; (ii) the mitochondria divide by means of the contraction of both outer and inner membranes. The forming furrow divides the "parental" mitochondrion into two equal "daughter" parts; (iii) at the four-cell stage the division ceases, and mitochondria start to grow, so that the mitochondrial length increases; (iv) cell differentiation further stimulates elongation of rod-shaped mitochondria, and the ratio of rod-shaped to spherical mitochondria changes; (v) in an unfertilised egg, the mitochondria are in a condensed form; after fertilisation all the mitochondria acquire a conventional form. Modern concepts of chondriome proliferation in eukaryotic cells are discussed. PMID:11699864

  13. 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

  14. A New, Dynamic Era for Somatic Cell Nuclear Transfer?

    PubMed

    Loi, Pasqualino; Iuso, Domenico; Czernik, Marta; Ogura, Atsuo

    2016-10-01

    Cloning animals by somatic cell nuclear transfer (SCNT) has remained an uncontrollable process for many years. High rates of embryonic losses, stillbirths, and postnatal mortality have been typical outcomes. These developmental problems arise from abnormal genomic reprogramming: the capacity of the oocyte to reset the differentiated memory of a somatic cell. However, effective reprogramming strategies are now available. These target the whole genome or single domains such as the Xist gene, and their effectiveness has been validated with the ability of experimental animals to develop to term. Thus, SCNT has become a controllable process that can be used to 'rescue' endangered species, and for biomedical research such as therapeutic cloning and the isolation of induced pluripotent stem cells (iPSCs). PMID:27118511

  15. Gene-specific of endocannabinoid receptor 1 (cnr1a) by ethanol probably leads to the development of fetal alcohol spectrum disorder (FASD) phenotypes in Japanese rice fish (Oryzias latipes) embryogenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Developmental ethanol exposure is able to induce Fetal Alcohol Spectrum Disorder (FASD) phenotypes in Japanese rice fish (Oryzias latipes). This study investigated possible differential expression of cannabinoid receptor (cnr) mRNAs during Japanese rice fish embryogenesis and variability to ethanol-...

  16. G-protein-coupled estrogen receptor 1 is involved in brain development during zebrafish (Danio rerio) embryogenesis

    SciTech Connect

    Shi, Yanan; Liu, Xiaochun; Zhu, Pei; Li, Jianzhen; Sham, Kathy W.Y.; Cheng, Shuk Han; Li, Shuisheng; Zhang, Yong; Cheng, Christopher H.K.; Lin, Haoran

    2013-05-24

    Highlights: •The Gper expression was detected in the developing brain of zebrafish. •Gper morpholino knockdown induced apoptosis of brain cells. •Gper morpholino knockdown reduced expression in neuron markers. •Zebrafish Gper may be involved in neuronal development. -- Abstract: G-protein-coupled estrogen receptor 1 (Gper, formerly known as GPR30) is found to be a trophic and protective factor in mediating action of estrogen in adult brain, while its role in developing brain remains to be elucidated. Here we present the expression pattern of Gper and its functions during embryogenesis in zebrafish. Both the mRNA and protein of Gper were detected throughout embryogenesis. Whole mount in situ hybridization (WISH) revealed a wide distribution of gper mRNAs in various regions of the developing brain. Gper knockdown by specific morpholinos resulted in growth retardation in embryos and morphological defects in the developing brain. In addition, induced apoptosis, decreased proliferation of the brain cells and maldevelopment of sensory and motor neurons were also found in the morphants. Our results provide novel insights into Gper functions in the developing brain, revealing that Gper can maintain the survival of the brain cells, and formation and/or differentiation of the sensory and motor neurons.

  17. Cell-of-Origin-Specific 3D Genome Structure Acquired during Somatic Cell Reprogramming

    PubMed Central

    Krijger, Peter Hugo Lodewijk; Di Stefano, Bruno; de Wit, Elzo; Limone, Francesco; van Oevelen, Chris; de Laat, Wouter; Graf, Thomas

    2016-01-01

    Summary Forced expression of reprogramming factors can convert somatic cells into induced pluripotent stem cells (iPSCs). Here we studied genome topology dynamics during reprogramming of different somatic cell types with highly distinct genome conformations. We find large-scale topologically associated domain (TAD) repositioning and alterations of tissue-restricted genomic neighborhoods and chromatin loops, effectively erasing the somatic-cell-specific genome structures while establishing an embryonic stem-cell-like 3D genome. Yet, early passage iPSCs carry topological hallmarks that enable recognition of their cell of origin. These hallmarks are not remnants of somatic chromosome topologies. Instead, the distinguishing topological features are acquired during reprogramming, as we also find for cell-of-origin-dependent gene expression patterns. PMID:26971819

  18. Cell-of-Origin-Specific 3D Genome Structure Acquired during Somatic Cell Reprogramming.

    PubMed

    Krijger, Peter Hugo Lodewijk; Di Stefano, Bruno; de Wit, Elzo; Limone, Francesco; van Oevelen, Chris; de Laat, Wouter; Graf, Thomas

    2016-05-01

    Forced expression of reprogramming factors can convert somatic cells into induced pluripotent stem cells (iPSCs). Here we studied genome topology dynamics during reprogramming of different somatic cell types with highly distinct genome conformations. We find large-scale topologically associated domain (TAD) repositioning and alterations of tissue-restricted genomic neighborhoods and chromatin loops, effectively erasing the somatic-cell-specific genome structures while establishing an embryonic stem-cell-like 3D genome. Yet, early passage iPSCs carry topological hallmarks that enable recognition of their cell of origin. These hallmarks are not remnants of somatic chromosome topologies. Instead, the distinguishing topological features are acquired during reprogramming, as we also find for cell-of-origin-dependent gene expression patterns.

  19. Measurement and assessment of somatic symptoms.

    PubMed

    Chaturvedi, Santosh K; Desai, Geetha

    2013-02-01

    Somatic symptoms are common presentations in health settings. They can manifest as symptoms of another underlying mental disorder or be termed as medically unexplained. When they are medically unexplained they are invariably subsumed under the diagnostic categories of somatoform disorders. They are associated with interference in functioning, poor quality of life and are burdensome on health resources. The measurement of these symptoms is essential for understanding the individual and planning treatment. There are various instruments that have somatic symptoms measurement in their items. The tools have included somatic symptoms measurement in measuring general psychopathology, somatic symptoms as part of anxiety and depression, somatic symptoms specifically, and as a screening instrument for somatoform disorders. The advantages and disadvantages of common measures have been discussed. It appears that no one measure fulfils the essential criteria of an ideal measure for somatic symptoms. The measures of somatic symptoms should also be culturally sensitive and serve diagnostic, prognostic and heuristic purposes. These aspects are highlighted in the review.

  20. 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

  1. Analysis of chlorophyll fluorescence reveals stage specific patterns of chloroplast-containing cells during Arabidopsis embryogenesis.

    PubMed

    Tejos, Ricardo I; Mercado, Ana V; Meisel, Lee A

    2010-01-01

    The basic body plan of a plant is established early in embryogenesis when cells differentiate, giving rise to the apical and basal regions of the embryo. Using chlorophyll fluorescence as a marker for chloroplasts, we have detected specific patterns of chloroplast-containing cells at specific stages of embryogenesis. Non-randomly distributed chloroplast-containing cells are seen as early as the globular stage of embryogenesis in Arabidopsis. In the heart stage of embryogenesis, chloroplast containing cells are detected in epidermal cells as well as a central region of the heart stage embryo, forming a triangular septum of chloroplast-containing cells that divides the embryo into three equal sectors. Torpedo stage embryos have chloroplast-containing epidermal cells and a central band of chloroplast-containing cells in the cortex layer, just below the shoot apical meristem. In the walking-stick stage of embryogenesis, chloroplasts are present in the epidermal, cortex and endodermal cells. The chloroplasts appear reduced or absent from the provascular and columella cells of walking-stick stage embryos. These results suggest that there is a tight regulation of plastid differentiation during embryogenesis that generates specific patterns of chloroplast-containing cells in specific cell layers at specific stages of embryogenesis.

  2. 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

  3. [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

  4. 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?

  5. Mutagenic effects of some water-soluble metal compounds in a somatic eye-color test system in Drosophila melanogaster.

    PubMed

    Rasmuson, A

    1985-01-01

    Nickel, cadmium, lead, arsenic, manganese and chromium salts as well as MeHgOH were screened for mutagenicity, using a sensitive somatic eye-color test system in Drosophila melanogaster. The test is based on the insertion of a mobile element which causes instability in the white locus that is somatically enhanced by mutagens. This white locus expression is combined with a mutation, zeste, in another gene, to produce a light yellow eye color. Larval feeding with mutagens causes somatic mutations in the eye imaginal disc cells that develop into easily detectable red spots in the yellow eyes of adult males. Survival tests showed large differences in the toxicity of different metals, but only hexavalent chromium increased the frequency of somatic mutations above the control level. When combined treatments were carried out with MMS and various metals, sodium arsenite caused a reduction of the MMS-induced mutation frequency while methylmercury increased the frequency of somatic spots.

  6. Pilates, Mindfulness and Somatic Education.

    PubMed

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

    2013-12-01

    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

  7. Why do some psychiatric patients somatize?

    PubMed

    de León, J; Saiz-Ruiz, J; Chinchilla, A; Morales, P

    1987-08-01

    A study was made of a series of 139 outpatients referred by the medical and surgical services of a general hospital for evaluation by the psychiatry unit. In accordance with established criteria, this population was divided into somatizers (56) and non-somatizers (75), and the socio-demographic and clinical characteristics of both groups were comparatively analyzed. The results show that the group of somatizers was younger, had more histrionic personality traits and more stress factors related with alterations in interpersonal relationships or death or disease of relatives. It is emphasized that somatization is poorly known by psychiatrists--whose diagnostic criteria practically omit these aspects--and by other physicians, in spite of its importance and frequency. PMID:3673644

  8. Self-renewal and pluripotency acquired through somatic reprogramming to human cancer stem cells.

    PubMed

    Nagata, Shogo; Hirano, Kunio; Kanemori, Michele; Sun, Liang-Tso; Tada, Takashi

    2012-01-01

    Human induced pluripotent stem cells (iPSCs) are reprogrammed by transient expression of transcription factors in somatic cells. Approximately 1% of somatic cells can be reprogrammed into iPSCs, while the remaining somatic cells are differentially reprogrammed. Here, we established induced pluripotent cancer stem-like cells (iCSCs) as self-renewing pluripotent cell clones. Stable iCSC lines were established from unstable induced epithelial stem cell (iESC) lines through re-plating followed by embryoid body formation and serial transplantation. iCSCs shared the expression of pluripotent marker genes with iPSCs, except for REX1 and LIN28, while exhibited the expression of somatic marker genes EMP1 and PPARγ. iESCs and iCSCs could generate teratomas with high efficiency by implantation into immunodeficient mice. The second iCSCs isolated from dissociated cells of teratoma from the first iCSCs were stably maintained, showing a gene expression profile similar to the first iCSCs. In the first and second iCSCs, transgene-derived Oct4, Sox2, Klf4, and c-Myc were expressed. Comparative global gene expression analyses demonstrated that the first iCSCs were similar to iESCs, and clearly different from human iPSCs and somatic cells. In iCSCs, gene expression kinetics of the core pluripotency factor and the Myc-related factor were pluripotent type, whereas the polycomb complex factor was somatic type. These findings indicate that pluripotent tumorigenicity can be conferred on somatic cells through up-regulation of the core pluripotency and Myc-related factors, prior to establishment of the iPSC molecular network by full reprogramming through down-regulation of the polycomb complex factor.

  9. Reward and somatic changes during precipitated nicotine withdrawal in rats: centrally and peripherally mediated effects.

    PubMed

    Watkins, S S; Stinus, L; Koob, G F; Markou, A

    2000-03-01

    The negative affective aspects of nicotine withdrawal have been hypothesized to contribute to tobacco dependence. In the present studies in rats, brain stimulation reward thresholds, conditioned place aversions, and somatic signs of withdrawal were used to investigate the role of central and peripheral nicotinic acetylcholine and opioid receptors in nicotine withdrawal. Rats prepared with s.c. osmotic mini-pumps delivering 9.0 mg/kg/day nicotine hydrogen tartrate or saline were administered various doses of the nicotinic antagonists mecamylamine (s.c.), chlorisondamine (s. c. or i.c.v.), dihydro-beta-erythroidine (s.c.), or the opiate antagonist naloxone (s.c.). Nicotine-treated rats receiving mecamylamine or i.c.v. chlorisondamine exhibited elevated thresholds and more somatic signs than saline-treated rats. Nicotine-treated rats receiving s.c. chlorisondamine, at doses that do not readily cross the blood-brain barrier, exhibited more somatic signs than saline-treated rats with no threshold elevations. Naloxone administration produced threshold elevations and somatic signs only at high doses that induced similar magnitude effects in both nicotine- and saline-treated subjects. Mecamylamine or dihydro-beta-erythroidine administration induced conditioned place aversions in nicotine-treated rats but required higher doses than those needed to precipitate threshold elevations. In contrast, naloxone administration induced conditioned place aversions at lower doses than those required to precipitate threshold elevations and somatic signs. These data provide evidence for a dissociation between centrally mediated elevations in reward thresholds and somatic signs that are both centrally and peripherally mediated. Furthermore, threshold elevations and somatic signs of withdrawal appear to be mediated by cholinergic neurotransmission, whereas conditioned place aversions appear to be primarily mediated by the opioid system.

  10. Progress in the reprogramming of somatic cells.

    PubMed

    Ma, Tianhua; Xie, Min; Laurent, Timothy; Ding, Sheng

    2013-02-01

    Pluripotent stem cells can differentiate into nearly all types of cells in the body. This unique potential provides significant promise for cell-based therapies to restore tissues or organs destroyed by injuries, degenerative diseases, aging, or cancer. The discovery of induced pluripotent stem cell (iPSC) technology offers a possible strategy to generate patient-specific pluripotent stem cells. However, because of concerns about the specificity, efficiency, kinetics, and safety of iPSC reprogramming, improvements or fundamental changes in this process are required before their effective clinical use. A chemical approach is regarded as a promising strategy to improve and change the iPSC process. Dozens of small molecules have been identified that can functionally replace reprogramming factors and significantly improve iPSC reprogramming. In addition to the prospect of deriving patient-specific tissues and organs from iPSCs, another attractive strategy for regenerative medicine is transdifferentiation-the direct conversion of one somatic cell type to another. Recent studies revealed a new paradigm of transdifferentiation: using transcription factors used in iPSC generation to induce transdifferentiation or called iPSC transcription factor-based transdifferentiation. This type of transdifferentiation not only reveals and uses the developmentally plastic intermediates generated during iPSC reprogramming but also produces a wide range of cells, including expandable tissue-specific precursor cells. Here, we review recent progress of small molecule approaches in the generation of iPSCs. In addition, we summarize the new concept of iPSC transcription factor-based transdifferentiation and discuss its application in generating various lineage-specific cells, especially cardiovascular cells.

  11. 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)

  12. Human somatic cell nuclear transfer and cloning.

    PubMed

    2012-10-01

    This document presents arguments that conclude that it is unethical to use somatic cell nuclear transfer (SCNT) for infertility treatment due to concerns about safety; the unknown impact of SCNT on children, families, and society; and the availability of other ethically acceptable means of assisted reproduction. This document replaces the ASRM Ethics Committee report titled, "Human somatic cell nuclear transfer (cloning)," last published in Fertil Steril 2000;74:873-6.

  13. (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.

  14. Regenerative therapy for neuronal diseases with transplantation of somatic stem cells.

    PubMed

    Kanno, Hiroshi

    2013-10-26

    Pluripotent stem cells, which are capable of differentiating in various species of cells, are hoped to be donor cells in transplantation in regenerative medicine. Embryonic stem (ES) cells and induced pluripotent stem cells have the potential to differentiate in approximately all species of cells. However, the proliferating ability of these cells is high and the cancer formation ability is also recognized. In addition, ethical problems exist in using ES cells. Somatic stem cells with the ability to differentiate in various species of cells have been used as donor cells for neuronal diseases, such as amyotrophic lateral sclerosis, spinal cord injury, Alzheimer disease, cerebral infarction and congenital neuronal diseases. Human mesenchymal stem cells derived from bone marrow, adipose tissue, dermal tissue, umbilical cord blood and placenta are usually used for intractable neuronal diseases as somatic stem cells, while neural progenitor/stem cells and retinal progenitor/stem cells are used for a few congenital neuronal diseases and retinal degenerative disease, respectively. However, non-treated somatic stem cells seldom differentiate to neural cells in recipient neural tissue. Therefore, the contribution to neuronal regeneration using non-treated somatic stem cells has been poor and various differential trials, such as the addition of neurotrophic factors, gene transfer, peptide transfer for neuronal differentiation of somatic stem cells, have been performed. Here, the recent progress of regenerative therapies using various somatic stem cells is described.

  15. Multicellularity makes somatic differentiation evolutionarily stable

    PubMed Central

    Wahl, Mary E.; Murray, Andrew W.

    2016-01-01

    Many multicellular organisms produce two cell lineages: germ cells, whose descendants produce the next generation, and somatic cells, which support, protect, and disperse the germ cells. This germ-soma demarcation has evolved independently in dozens of multicellular taxa but is absent in unicellular species. A common explanation holds that in these organisms, inefficient intercellular nutrient exchange compels the fitness cost of producing nonreproductive somatic cells to outweigh any potential benefits. We propose instead that the absence of unicellular, soma-producing populations reflects their susceptibility to invasion by nondifferentiating mutants that ultimately eradicate the soma-producing lineage. We argue that multicellularity can prevent the victory of such mutants by giving germ cells preferential access to the benefits conferred by somatic cells. The absence of natural unicellular, soma-producing species previously prevented these hypotheses from being directly tested in vivo: to overcome this obstacle, we engineered strains of the budding yeast Saccharomyces cerevisiae that differ only in the presence or absence of multicellularity and somatic differentiation, permitting direct comparisons between organisms with different lifestyles. Our strains implement the essential features of irreversible conversion from germ line to soma, reproductive division of labor, and clonal multicellularity while maintaining sufficient generality to permit broad extension of our conclusions. Our somatic cells can provide fitness benefits that exceed the reproductive costs of their production, even in unicellular strains. We find that nondifferentiating mutants overtake unicellular populations but are outcompeted by multicellular, soma-producing strains, suggesting that multicellularity confers evolutionary stability to somatic differentiation. PMID:27402737

  16. Multicellularity makes somatic differentiation evolutionarily stable.

    PubMed

    Wahl, Mary E; Murray, Andrew W

    2016-07-26

    Many multicellular organisms produce two cell lineages: germ cells, whose descendants produce the next generation, and somatic cells, which support, protect, and disperse the germ cells. This germ-soma demarcation has evolved independently in dozens of multicellular taxa but is absent in unicellular species. A common explanation holds that in these organisms, inefficient intercellular nutrient exchange compels the fitness cost of producing nonreproductive somatic cells to outweigh any potential benefits. We propose instead that the absence of unicellular, soma-producing populations reflects their susceptibility to invasion by nondifferentiating mutants that ultimately eradicate the soma-producing lineage. We argue that multicellularity can prevent the victory of such mutants by giving germ cells preferential access to the benefits conferred by somatic cells. The absence of natural unicellular, soma-producing species previously prevented these hypotheses from being directly tested in vivo: to overcome this obstacle, we engineered strains of the budding yeast Saccharomyces cerevisiae that differ only in the presence or absence of multicellularity and somatic differentiation, permitting direct comparisons between organisms with different lifestyles. Our strains implement the essential features of irreversible conversion from germ line to soma, reproductive division of labor, and clonal multicellularity while maintaining sufficient generality to permit broad extension of our conclusions. Our somatic cells can provide fitness benefits that exceed the reproductive costs of their production, even in unicellular strains. We find that nondifferentiating mutants overtake unicellular populations but are outcompeted by multicellular, soma-producing strains, suggesting that multicellularity confers evolutionary stability to somatic differentiation. PMID:27402737

  17. 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

  18. Overexpression of Late Embryogenesis Abundant 14 enhances Arabidopsis salt stress tolerance.

    PubMed

    Jia, Fengjuan; Qi, Shengdong; Li, Hui; Liu, Pu; Li, Pengcheng; Wu, Changai; Zheng, Chengchao; Huang, Jinguang

    2014-11-28

    Late embryogenesis abundant (LEA) proteins are implicated in various abiotic stresses in higher plants. In this study, we identified a LEA protein from Arabidopsis thaliana, AtLEA14, which was ubiquitously expressed in different tissues and remarkably induced with increased duration of salt treatment. Subcellular distribution analysis demonstrated that AtLEA14 was mainly localized in the cytoplasm. Transgenic Arabidopsis and yeast overexpressing AtLEA14 all exhibited enhanced tolerance to high salinity. The transcripts of salt stress-responsive marker genes (COR15a, KIN1, RD29B and ERD10) were overactivated in AtLEA14 overexpressing lines compared with those in wild type plants under normal or salt stress conditions. In vivo and in vitro analysis showed that AtLEA14 could effectively stabilize AtPP2-B11, an important E3 ligase. These results suggested that AtLEA14 had important protective functions under salt stress conditions in Arabidopsis. PMID:25450686

  19. Overexpression of Late Embryogenesis Abundant 14 enhances Arabidopsis salt stress tolerance

    SciTech Connect

    Jia, Fengjuan Qi, Shengdong Li, Hui Liu, Pu Li, Pengcheng Wu, Changai Zheng, Chengchao Huang, Jinguang

    2014-11-28

    Highlights: • It is the first time to investigate the biological function of AtLEA14 in salt stress response. • AtLEA14 enhances the salt stress tolerance both in Arabidopsis and yeast. • AtLEA14 responses to salt stress by stabilizing AtPP2-B11, an E3 ligase, under normal or salt stress conditions. - Abstract: Late embryogenesis abundant (LEA) proteins are implicated in various abiotic stresses in higher plants. In this study, we identified a LEA protein from Arabidopsis thaliana, AtLEA14, which was ubiquitously expressed in different tissues and remarkably induced with increased duration of salt treatment. Subcellular distribution analysis demonstrated that AtLEA14 was mainly localized in the cytoplasm. Transgenic Arabidopsis and yeast overexpressing AtLEA14 all exhibited enhanced tolerance to high salinity. The transcripts of salt stress-responsive marker genes (COR15a, KIN1, RD29B and ERD10) were overactivated in AtLEA14 overexpressing lines compared with those in wild type plants under normal or salt stress conditions. In vivo and in vitro analysis showed that AtLEA14 could effectively stabilize AtPP2-B11, an important E3 ligase. These results suggested that AtLEA14 had important protective functions under salt stress conditions in Arabidopsis.

  20. 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

  1. Somatic Mutations Are Not Observed by Exome Sequencing of Lymphocyte DNA from Monozygotic Twins Discordant for Congenital Hypothyroidism due to Thyroid Dysgenesis

    PubMed Central

    Magne, Fabien; Serpa, Roman; Van Vliet, Guy; Samuels, Mark E.; Deladoëy, Johnny

    2016-01-01

    Background/Aims Congenital primary hypothyroidism (CH) is a rare pediatric disorder estimated to occur in about 1: 2,500 live births. Approximately half of these cases entail ectopic thyroid tissue, which is believed to result from a migration defect during embryogenesis. Approximately 3% of CH cases are explained by mutation(s) in known genes, most of which are transcription factors implicated in the embryology of the thyroid gland. Surprisingly, monozygotic (MZ) twins are usually discordant for CH due to thyroid dysgenesis, suggesting that most cases are not caused by transmitted genetic variation. One possible explanation is somatic mutation in genes involved in thyroid migration occurring after zygotic twinning. Such mutations should be observed only in the affected twin. Methods To test the hypothesis of somatic mutation, we performed whole exome sequencing of DNA from three pairs of MZ twins discordant for CH with ectopic glands. Results We found no somatic mutations exclusive to any of the three affected twins or in any of the unaffected twins. Conclusion Either somatic mutations are not significant for the etiology of CH or else such mutations lie outside regions of the genome accessible by exome sequencing technology. PMID:25277881

  2. Replication of somatic micronuclei in bovine enucleated oocytes

    PubMed Central

    2012-01-01

    Background Microcell-mediated chromosome transfer (MMCT) was developed to introduce a low number of chromosomes into a host cell. We have designed a novel technique combining part of MMCT with somatic cell nuclear transfer, which consists of injecting a somatic micronucleus into an enucleated oocyte, and inducing its cellular machinery to replicate such micronucleus. It would allow the isolation and manipulation of a single or a low number of somatic chromosomes. Methods Micronuclei from adult bovine fibroblasts were produced by incubation in 0.05 μg/ml demecolcine for 46 h followed by 2 mg/ml mitomycin for 2 h. Cells were finally treated with 10 μg/ml cytochalasin B for 1 h. In vitro matured bovine oocytes were mechanically enucleated and intracytoplasmatically injected with one somatic micronucleus, which had been previously exposed [Micronucleus- injected (+)] or not [Micronucleus- injected (−)] to a transgene (50 ng/μl pCX-EGFP) during 5 min. Enucleated oocytes [Enucleated (+)] and parthenogenetic [Parthenogenetic (+)] controls were injected into the cytoplasm with less than 10 pl of PVP containing 50 ng/μl pCX-EGFP. A non-injected parthenogenetic control [Parthenogenetic (−)] was also included. Two hours after injection, oocytes and reconstituted embryos were activated by incubation in 5 μM ionomycin for 4 min + 1.9 mM 6-DMAP for 3 h. Cleavage stage and egfp expression were evaluated. DNA replication was confirmed by DAPI staining. On day 2, Micronucleus- injected (−), Parthenogenetic (−) and in vitro fertilized (IVF) embryos were karyotyped. Differences among treatments were determined by Fisher′s exact test (p≤0.05). Results All the experimental groups underwent the first cell divisions. Interestingly, a low number of Micronucleus-injected embryos showed egfp expression. DAPI staining confirmed replication of micronuclei in most of the evaluated embryos. Karyotype analysis revealed that all Micronucleus-injected embryos had fewer than 15

  3. 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.

  4. 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.

  5. A dynamic role for sterols in embryogenesis of Pisum sativum.

    PubMed

    Schrick, Kathrin; Cordova, Cindy; Li, Grace; Murray, Leigh; Fujioka, Shozo

    2011-04-01

    Molecular roles of sterols in plant development remain to be elucidated. To investigate sterol composition during embryogenesis, the occurrence of 25 steroid compounds in stages of developing seeds and pods of Pisum sativum was examined by GC-MS analysis. Immature seeds containing very young embryos exhibited the greatest concentrations of sterols. Regression models indicated that the natural log of seed or pod fr. wt was a consistent predictor of declining sterol content during embryonic development. Although total sterol levels were reduced in mature embryos, the composition of major sterols sitosterol and campesterol remained relatively constant in all 12 seed stages examined. In mature seeds, a significant decrease in isofucosterol was observed, as well as minor changes such as increases in cycloartenol branch sterols and campesterol derivatives. In comparison to seeds and pods, striking differences in composition were observed in sterol profiles of stems, shoots, leaves, flowers and flower buds, as well as cotyledons versus radicles. The highest levels of isofucosterol, a precursor to sitosterol, occurred in young seeds and flower buds, tissues that contain rapidly dividing cells and cells undergoing differentiation. Conversely, the highest levels of stigmasterol, a derivative of sitosterol, were found in fully-differentiated leaves while all seed stages exhibited low levels of stigmasterol. The observed differences in sterol content were correlated to mRNA expression data for sterol biosynthesis genes from Arabidopsis. These findings implicate the coordinated expression of sterol biosynthesis enzymes in gene regulatory networks underlying the embryonic development of flowering plants.

  6. Localization of dystrophin gene transcripts during mouse embryogenesis

    PubMed Central

    1992-01-01

    The spatial and temporal expression of the dystrophin gene has been examined during mouse embryogenesis, using in situ hybridization on tissue sections with a probe from the 5' end of the dystrophin coding sequence. In striated muscle, dystrophin transcripts are detectable from about 9 d in the heart and slightly later in skeletal muscle. However, there is an important difference between the two types of muscle: the heart is already functional as a contractile organ before the appearance of dystrophin transcripts, whereas this is not the case in skeletal muscle, where dystrophin and myosin heavy chain transcripts are first detectable at the same time. In the heart, dystrophin transcripts accumulate initially in the outflow tract and, at later stages, in both the atria and ventricles. In skeletal muscle, the gene is expressed in all myocytes irrespective of fiber type. In smooth muscle dystrophin transcripts are first detectable from 11 d post coitum in blood vessels, and subsequently in lung bronchi and in the digestive tract. The other major tissue where the dystrophin gene is expressed is the brain, where transcripts are clearly detectable in the cerebellum from 13 d. High-level expression of the gene is also seen in particular regions of the forebrain involved in the regulation of circadian rhythms, the endocrine system, and olfactory function, not previously identified in this context. The findings are discussed in the context of the pathology of Duchenne muscular dystrophy. PMID:1429837

  7. 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.

  8. Chitin oligosaccharides as candidate patterning agents in zebrafish embryogenesis.

    PubMed

    Semino, C E; Allende, M L

    2000-02-01

    In this work we investigate the possible function of N-acetyl-chitooligosaccharides (NACOs) produced during zebrafish (Danio rerio) development. First, we show that NACOs are synthesized in vivo during early embryogenesis in the zebrafish. Second, we demonstrate that injection of a pure bacterial chitinase into one-cell stage embryos elicits developmental defects in which the posterior trunk and tail of developing embryo are severely affected. In addition, an endogenous chitinase activity detected both intra- and extracellularly is described, suggesting that cells may secrete it into the extracellular space. Moreover, this compartmentalization appears to be functionally relevant as inhibition of the extracellular, but not the intracellular, endogenous chitinase activity causes morphological defects similar to those seen in embryos injected with chitinase 63. Finally, analysis of the expression of the zebrafish ZDG42 gene, which has been suggested to be involved in synthesis of NACOs, is described. Transcripts are detected from late blastula stage, during gastrulation, and move as an anterior-posterior wave of expression in adaxial mesoderm during somitogenesis.

  9. Epidermal patterning genes are active during embryogenesis in Arabidopsis.

    PubMed

    Costa, Silvia; Dolan, Liam

    2003-07-01

    Epidermal cells in the root of Arabidopsis seedling differentiate either as hair or non-hair cells, while in the hypocotyl they become either stomatal or elongated cells. WEREWOLF (WER) and GLABRA2 (GL2) are positive regulators of non-hair and elongated cell development. CAPRICE (CPC) is a positive regulator of hair cell development in the root. We show that WER, GL2 and CPC are expressed and active during the stages of embryogenesis when the pattern of cells in the epidermis of the root-hypocotyl axis forms. GL2 is first expressed in the future epidermis in the heart stage embryo and its expression is progressively restricted to those cells that will acquire a non-hair identity in the transition between torpedo and mature stage. The expression of GL2 at the heart stage requires WER function. WER and CPC are transiently expressed throughout the root epidermal layer in the torpedo stage embryo when the cell-specific pattern of GL2 expression is being established in the epidermis. We also show that WER positively regulates CPC transcription and GL2 negatively regulates WER transcription in the mature embryo. We propose that the restriction of GL2 to the future non-hair cells in the root epidermis can be correlated with the activities of WER and CPC during torpedo stage. In the embryonic hypocotyl we show that WER controls GL2 expression. We also provide evidence indicating that CPC may also regulate GL2 expression in the hypocotyl.

  10. Epicardial-Derived Adrenomedullin Drives Cardiac Hyperplasia During Embryogenesis

    PubMed Central

    Wetzel-Strong, Sarah E.; Li, Manyu; Klein, Klara R.; Nishikimi, Toshio; Caron, Kathleen M.

    2014-01-01

    Background Growth promoting signals from the epicardium are essential for driving myocardial proliferation during embryogenesis. In adults, these signals become reactivated following injury and promote angiogenesis and myocardial repair. Therefore, identification of such paracrine factors could lead to novel therapeutic strategies. The multi-functional peptide adrenomedullin (Adm = gene, AM = protein) is required for normal heart development. Moreover, elevated plasma AM following myocardial infarction offers beneficial cardioprotection and serves as a powerful diagnostic and prognostic indication of disease severity. Results Here, we developed a new model of Adm overexpression by stabilizing the Adm mRNA through gene-targeted replacement of the endogenous 3′ untranslated region. As expected, Admhi/hi mice express three-times more AM than controls in multiple tissues, including the heart. Despite normal blood pressures, Admhi/hi mice unexpectedly showed significantly enlarged hearts due to increased cardiac hyperplasia during development. The targeting vector was designed to allow for reversion to wild-type levels by means of Cre-mediated modification. Using this approach, we demonstrate that AM derived from the epicardium, but not the myocardium or cardiac fibroblast, is responsible for driving cardiomyocyte hyperplasia. Conclusions AM is produced by the epicardium and drives myocyte proliferation during development, thus representing a novel and clinically relevant factor potentially related to mechanisms of cardiac repair after injury. PMID:24123312

  11. Somatic markers, working memory, and decision making.

    PubMed

    Hinson, John M; Jameson, Tina L; Whitney, Paul

    2002-12-01

    The somatic marker hypothesis formulated by Damasio (e.g., 1994; Damasio, Tranel, & Damasio, 1991) argues that affective reactions ordinarily guide and simplify decision making. Although originally intended to explain decision-making deficits in people with specific frontal lobe damage, the hypothesis also applies to decision-making problems in populations without brain injury. Subsequently, the gambling task was developed by Bechara (Bechara, Damasio, Damasio, & Anderson, 1994) as a diagnostic test of decision-making deficit in neurological populations. More recently, the gambling task has been used to explore implications of the somatic marker hypothesis, as well as to study suboptimal decision making in a variety of domains. We examined relations among gambling task decision making, working memory (WM) load, and somatic markers in a modified version of the gambling task. Increased WM load produced by secondary tasks led to poorer gambling performance. Declines in gambling performance were associated with the absence of the affective reactions that anticipate choice outcomes and guide future decision making. Our experiments provide evidence that WM processes contribute to the development of somatic markers. If WM functioning is taxed, somatic markers may not develop, and decision making may thereby suffer. PMID:12641178

  12. GA3 stimulates the formation and germination of somatic embryos and the expression of a KNOTTED-like homeobox gene of Cocos nucifera (L.).

    PubMed

    Montero-Córtes, M; Sáenz, Luis; Córdova, I; Quiroz, A; Verdeil, J-L; Oropeza, C

    2010-09-01

    The micropropagation of coconut palm has progressed rapidly; yet, there are constraints with regard to the number of somatic embryos formed and their germination. To overcome these, we tested the effect of gibberellic acid and characterized genes of the KNOX family. Gibberellic acid at 0.5 muM increased 1.5-fold the number of calli forming somatic embryos and twofold the number of somatic embryos per callus, calli with germinating embryos and the number of germinating somatic embryos per callus. With regard to the study of KNOX family genes, the complete sequences of two KNOX-like genes were obtained for CnKNOX1 and CnKNOX2. The deduced amino acid sequence of both showed highly conserved domains characteristic of KNOX genes. CnKNOX1 showed high homology with KNOX class I proteins. CnKNOX1 expression was detected throughout the embryogenesis process except in somatic embryos at the pro-globular stage, and was highest in somatic embryos at the coleoptilar stage. No detection of CnKNOX1 expression occurred in calli with aberrant embryos. The addition of gibberellic acid stimulated the expression of CnKNOX1 earlier and the relative expression at all stages was higher. CnKNOX2 expression occurred at all stages peaking at the globular stage, but gibberellic acid treatment decreased the expression. Gene expression was also analyzed in tissues of different organs of adult palms. With CnKNOX1, high level of expression was found in tissues of organs with, but not in those without, meristem, whereas CnKNOX2 expression was detected in tissues with and also in those without meristem. PMID:20582418

  13. Genetic and epigenetic changes in somatic hybrid introgression lines between wheat and tall wheatgrass

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Broad phenotypic variations were induced in derivatives of an asymmetric somatic hybridization of bread wheat (Triticum aestivum) and tall wheatgrass (Thinopyrum ponticum Podp); however, how did these variations happened was unknown. We explored the nature of these variations by cytogenetic assays ...

  14. 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

  15. Effect of different Thai traditional processing of various hot chili peppers on urethane-induced somatic mutation and recombination in Drosophila melanogaster: assessment of the role of glutathione transferase activity.

    PubMed

    Laohavechvanich, P; Kangsadalampai, K; Tirawanchai, N; Ketterman, A J

    2006-08-01

    Four different Thai traditional chili peppers, namely bird pepper (Capsicum frutescens), red chili spur peppers (Capsicum annuum), green bell peppers and sweet pepper (C. annuum) were investigated for their antimutagenic properties. Each chili was prepared in three formulations commonly used for chili food processing; raw paste (chili ground in water), pickled in vinegar or stir-fried in palm oil. Each sample was tested for its antimutagenic effect against urethane by using the somatic mutation and recombination of wing hair of Drosophila melanogaster as an indicator. Three-day-old larvae, trans-heterozygous for two genetic markers, multiple wing hairs mwh and orrigon (ORR;flr3), were exposed to urethane alone or in combination with each chili formulation. The various processing methods for chilies differentially extracted the antimutagenic chili components. The specific chili as well as the method of processing influenced the observed antimutagenic properties against urethane. This suggested each chili contains a unique complex mixture of many antimutagens. Co-treatment and pre-treatment experiments showed that both direct and indirect protective mechanisms are involved in an 'activation' process to give antimutagenesis effects. An association between antigenotoxicity and glutathione transferase activity could not be established.

  16. Altered A-to-I RNA Editing in Human Embryogenesis

    PubMed Central

    Mandel, Rachel; Ziskind, Anna; Nahor, Irit; Safran, Michal; Osenberg, Sivan; Sherf, Ofra; Rechavi, Gideon; Itskovitz-Eldor, Joseph

    2012-01-01

    Post-transcriptional events play an important role in human development. The question arises as to whether Adenosine to Inosine RNA editing, catalyzed by the ADAR (Adenosine Deaminase acting on RNA) enzymes, differs in human embryogenesis and in adulthood. We tested the editing of various target genes in coding (FLNA, BLCAP, CYFIP2) and non-coding sequences at their Alu elements (BRCA1, CARD11, RBBP9, MDM4, FNACC), as well as the transcriptional levels of the ADAR1 enzymes. This analysis was performed on five fetal and adult human tissues: brain, heart, liver, kidney, and spleen, as well as on human embryonic stem cells (hESCs), which represent the blastocyst stage in early human development. Our results show substantially greater editing activity for most adult tissue samples relative to fetal ones, in six of the eight genes tested. To test the effect of reduced A-to-I RNA editing activity in early human development we used human embryonic stem cells (hESCs) as a model and tried to generate hESC clones that overexpress the ADAR1–p110 isoform. We were unable to achieve overexpression of ADAR1–p110 by either transfection or lentiviral infection, though we easily generated hESC clones that expressed the GFP transgene and overexpressed ADAR1-p110 in 293T cells and in primary human foreskin fibroblast (HFF) cells. Moreover, in contrast to the expected overexpression of ADAR1-p110 protein following its introduction into hESCs, the expression levels of this protein decreased dramatically 24–48 hr post infection. Similar results were obtained when we tried to overexpress ADAR1-p110 in pluripotent embryonal carcinoma cells. This suggests that ADAR1 protein is substantially regulated in undifferentiated pluripotent hESCs. Overall, our data suggest that A-to-I RNA editing plays a critical role during early human development. PMID:22859999

  17. Cloning and expression of embryogenesis-regulating genes in Araucaria angustifolia (Bert.) O. Kuntze (Brazilian Pine)

    PubMed Central

    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

  18. The Requirement of WHIRLY1 for Embryogenesis Is Dependent on Genetic Background in Maize

    PubMed Central

    Zhang, Ya-Feng; Hou, Ming-Ming; Tan, Bao-Cai

    2013-01-01

    Plastid gene expression is essential to embryogenesis in higher plants, but the underlying mechanism is obscure. Through molecular characterization of an embryo defective 16 (emb16) locus, here we report that the requirement of plastid translation for embryogenesis is dependent on the genetic background in maize (Zea mays). The emb16 mutation arrests embryogenesis at transition stage and allows the endosperm to develop largely normally. Molecular cloning reveals that Emb16 encodes WHIRLY1 (WHY1), a DNA/RNA binding protein that is required for genome stability and ribosome formation in plastids. Interestingly, the previous why1 mutant alleles (why1-1 and why1-2) do not affect embryogenesis, only conditions albino seedlings. The emb16 allele of why1 mutation is in the W22 genetic background. Crosses between emb16 and why1-1 heterozygotes resulted in both defective embryos and albino seedlings in the F1 progeny. Introgression of the emb16 allele from W22 into A188, B73, Mo17, Oh51a and the why1-1 genetic backgrounds yielded both defective embryos and albino seedlings. Similar results were obtained with two other emb mutants (emb12 and emb14) that are impaired in plastid protein translation process. These results indicate that the requirement of plastid translation for embryogenesis is dependent on genetic backgrounds, implying a mechanism of embryo lethality suppression in maize. PMID:23840682

  19. Cloned ferrets produced by somatic cell nuclear transfer

    PubMed Central

    Li, Ziyi; Sun, Xingshen; Chen, Juan; Liu, Xiaoming; Wisely, Samantha M.; Zhou, Qi; Renard, Jean-Paul; Leno, Gregory H.; Engelhardt, John F.

    2007-01-01

    Somatic cell nuclear transfer (SCNT) offers great potential for developing better animal models of human disease. The domestic ferret (Mustela putorius furo) is an ideal animal model for influenza infections and potentially other human respiratory diseases such as cystic fibrosis, where mouse models have failed to reproduce the human disease phenotype. Here, we report the successful production of live cloned, reproductively competent, ferrets using species-specific SCNT methodologies. Critical to developing a successful SCNT protocol for the ferret was the finding that hormonal treatment, normally used for superovulation, adversely affected the developmental potential of recipient oocytes. The onset of Oct4 expression was delayed and incomplete in parthenogenetically activated oocytes collected from hormone-treated females relative to oocytes collected from females naturally mated with vasectomized males. Stimulation induced by mating and in vitro oocyte maturation produced the optimal oocyte recipient for SCNT. Although nuclear injection and cell fusion produced mid-term fetuses at equivalent rates (~3–4%), only cell fusion gave rise to healthy surviving clones. Single cell fusion rates and the efficiency of SCNT were also enhanced by placing two somatic cells into the perivitelline space. These species-specific modifications facilitated the birth of live, healthy, and fertile cloned ferrets. The development of microsatellite genotyping for domestic ferrets confirmed that ferret clones were genetically derived from their respective somatic cells and unrelated to their surrogate mother. With this technology, it is now feasible to begin generating genetically defined ferrets for studying transmissible and inherited human lung diseases. Cloning of the domestic ferret may also aid in recovery and conservation of the endangered black-footed ferret and European mink. PMID:16584722

  20. Glycoalkaloids as biomarkers for recognition of cultivated, wild, and somatic hybrids of potato.

    PubMed

    Savarese, Salvatore; Andolfi, Anna; Cimmino, Alessio; Carputo, Domenico; Frusciante, Luigi; Evidente, Antonio

    2009-04-01

    Cultivated and wild potato species synthesize a wide variety of steroidal glycoalkaloids (GAs). During breeding programs, species genomes are often put together through either sexual or somatic hybridization. Therefore, the determination of the GA composition of hybrids is very important in that it may affect either human consumption, or resistance to pathogen and pests. Here, we report the results of GA analysis performed on wild Solanum bulbocastanum, haploids of cultivated potato S. tuberosum and their interspecific somatic hybrids. GAs were extracted from tubers and analyzed by HPLC. HPLC Profile of S. tuberosum haploids showed, as expected, the presence of alpha-solanine and alpha-chaconine. The profile of S. bulbocastanum extract showed lack of alpha-solanine and alpha-chaconine, and the presence of four GAs. The GA pattern of the somatic hybrids was the sum of their parents' profile. This represents a noteworthy tool for their unequivocal recognition. Interestingly, two hybrids produced not only GAs of both parents but also new compounds to be further investigated. This provided evidence that somatic hybridization induced the synthesis of new metabolites. The nature of the probable unidentified GAs associated to S. bulbocastanum and its somatic hybrids was ascertained by chemical degradation and spectroscopic analysis of their aglycones and sugar moieties. Our results suggest their close relation with GAs of both wild and cultivated potato species.

  1. Decrease in antibody somatic hypermutation frequency under extreme, extended spaceflight conditions.

    PubMed

    Bascove, Matthieu; Guéguinou, Nathan; Schaerlinger, Bérénice; Gauquelin-Koch, Guillemette; Frippiat, Jean-Pol

    2011-09-01

    Somatic hypermutation diversifies antibody binding sites by introducing point mutations in the variable domains of rearranged immunoglobulin genes. In this study, we analyzed somatic hypermutation in variable heavy-chain (VH) domains of specific IgM antibodies of the urodele amphibian Pleurodeles waltl, immunized either on Earth or onboard the Mir space station. To detect somatic hypermutation, we aligned the variable domains of IgM heavy-chain transcripts with the corresponding VH gene. We also quantified NF-κB and activation-induced cytidine deaminase transcripts. Results were compared with those obtained using control animals immunized on Earth. Our data show that, as in most species of ectotherms, somatic hypermutation in P. waltl exhibits a mutational bias toward G and C bases. Furthermore, we show for the first time that somatic hypermutation occurs in space following immunization but at a lower frequency. This decrease is not due to a decrease in food intake or of the B-cell receptor/antigen interaction or to the absence of the germinal center-associated nuclear protein. It likely results from the combination of several spaceflight-associated changes, such as the severe reduction in T-cell activation, important perturbations of the cytoskeleton, and changes in the distribution of lymphocyte subpopulations and adhesion molecule expression.

  2. [Interdependance between somatic symptoms, sleep and dreams].

    PubMed

    Todorov, Assya

    2014-03-19

    Even in an established illness, somatic complains can hide other emotional inquiries. The therapist, always with a kind attitude, can ask more about patient's sexual life. This can be use of having a better idea of patient's life and problems. Talking about dreams can also be useful: it gives new and surprising elements about patient's personality and helps to progress on healing's way.

  3. 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…

  4. Writing Bodies: Somatic Mind in Composition Studies.

    ERIC Educational Resources Information Center

    Fleckenstein, Kristie S.

    1999-01-01

    Discusses the somatic mind, a permeable materiality in which mind and body resolve into a single entity which is (re)formed by the constantly shifting boundaries of discursive and corporeal intertextualities. Addresses its importance in composition studies. Critiques the poststructuralist disregard of corporeality. (CR)

  5. Somatic Disorders of Childhood and Adolescence.

    ERIC Educational Resources Information Center

    Siegel, Lawrence J.

    1990-01-01

    Briefly reviews number of theories which address role of psychological factors in etiology of somatic disorders. Focuses on psychological treatment approaches that have been used to alleviate or reduce symptomatic behaviors associated with eating disorders, elimination disorders, and headaches in children. Discusses role of school psychologists in…

  6. Sexual Abuse: Somatic and Emotional Reactions.

    ERIC Educational Resources Information Center

    Rimza, Mary Ellen; And Others

    1988-01-01

    Chart reviews and telephone interviews with 72 sexual abuse victims found that 48 of the children had symptoms similar to the "rape trauma" syndrome. Two-thirds of victims commonly had somatic complaints (such as abdominal pain) and emotional/behavioral problems (runaway behavior, suicide attempts). (DB)

  7. Genetic improvement of mastitis through selection on somatic cell count.

    PubMed

    Shook, G E

    1993-11-01

    Heredity influences both clinical mastitis and somatic cell score. Intramammary infection is the major cause of elevated somatic cell score. A nationwide program of genetic evaluation of dairy cattle for somatic cell score is being developed. Proper selection of artificial insemination sires, considering their genetic merit for both milk production and somatic cell score, will reduce the genetic increase in mastitis susceptibility that accompanies selection for high production. PMID:8242460

  8. Embryogenesis and plant regeneration from isolated microspores of Brassica rapa L. ssp. Oleifera.

    PubMed

    Burnett, L; Yarrow, S; Huang, B

    1992-05-01

    Conditions favourable to embryogenesis from isolated microspores of Brassica rapa L. ssp. oleifera (canola quality) were identified. A population with enhanced responsiveness for microspore embryogenesis (C200) was synthesized by crossing individual plants showing microspore embryogenic potential. For optimal microspore embryogenesis, buds (2-3mm in length, containing mid-late uninucieate microspores) were collected from older plants (2 months old) and microspores isolated and washed in iron-free B5 medium. NLN medium with its iron content reduced to half was beneficial for initial microspore culture. An elevated temperature(33-35°C) during the first day of culture, followed by maintenance at 25°C resulted in dozens of embryos from each isolation (about 100 buds). Seeds were obtained from plants regenerated from microsporederived embryos after colchicine treatment.

  9. Two HSP90 genes in mandarin fish Siniperca chuatsi: identification, characterization and their specific expression profiles during embryogenesis and under stresses.

    PubMed

    Wang, Peng-Fei; Zeng, Shuang; Xu, Peng; Zhou, Lei; Li, Gui-Feng

    2016-08-01

    HSP90 plays important roles in multiple cellular stress responses. Here, two cytoplasmic HSP90 isoforms, ScHSP90α and ScHSP90β, were identified from Siniperca chuatsi. Their cDNA and gDNA structures, amino acid sequence features, and sequence identities and phylogenetic analysis with other species were described. Their expression profiles during embryonic development in different tissues and under stressful conditions were analyzed using real-time quantitative PCR. During embryogenesis, transcripts of both genes were detected at low levels during the early developmental stages and were up-regulated from appearance of myomere for ScHSP90a and closure of blastopore for ScHSP90β. ScHSP90α showed a tissue-specific variation with high expression in ovary and brain under non-stressed conditions, while ScHSP90β was ubiquitously highly expressed in different tissues. Acute heat shock resulted in a strong up-regulation of ScHSP90α in heart, liver, and head kidney, while it only weakly induced ScHSP90β in these tissues. ScHSP90α was also markedly induced in liver in a time-dependent manner under hypoxia, while the expression of ScHSP90β was not affected by hypoxia. Additionally, Aeromonas hydrophila infection markedly augmented ScHSP90α in head kidney and spleen and mildly up-regulated ScHSP90β in spleen, while suppressing ScHSP90β in head kidney. These results suggest that ScHSP90α and ScHSP90β are differently involved in embryogenesis and under different environmental conditions including high temperature, hypoxia, and bacterial infection. This study will benefit to further clarify the roles of fish HSP90 isoforms in embryogenesis and under stressful conditions and contribute to further study on enhancing stress tolerance and disease resistance of mandarin fish. PMID:26820141

  10. Two HSP90 genes in mandarin fish Siniperca chuatsi: identification, characterization and their specific expression profiles during embryogenesis and under stresses.

    PubMed

    Wang, Peng-Fei; Zeng, Shuang; Xu, Peng; Zhou, Lei; Li, Gui-Feng

    2016-08-01

    HSP90 plays important roles in multiple cellular stress responses. Here, two cytoplasmic HSP90 isoforms, ScHSP90α and ScHSP90β, were identified from Siniperca chuatsi. Their cDNA and gDNA structures, amino acid sequence features, and sequence identities and phylogenetic analysis with other species were described. Their expression profiles during embryonic development in different tissues and under stressful conditions were analyzed using real-time quantitative PCR. During embryogenesis, transcripts of both genes were detected at low levels during the early developmental stages and were up-regulated from appearance of myomere for ScHSP90a and closure of blastopore for ScHSP90β. ScHSP90α showed a tissue-specific variation with high expression in ovary and brain under non-stressed conditions, while ScHSP90β was ubiquitously highly expressed in different tissues. Acute heat shock resulted in a strong up-regulation of ScHSP90α in heart, liver, and head kidney, while it only weakly induced ScHSP90β in these tissues. ScHSP90α was also markedly induced in liver in a time-dependent manner under hypoxia, while the expression of ScHSP90β was not affected by hypoxia. Additionally, Aeromonas hydrophila infection markedly augmented ScHSP90α in head kidney and spleen and mildly up-regulated ScHSP90β in spleen, while suppressing ScHSP90β in head kidney. These results suggest that ScHSP90α and ScHSP90β are differently involved in embryogenesis and under different environmental conditions including high temperature, hypoxia, and bacterial infection. This study will benefit to further clarify the roles of fish HSP90 isoforms in embryogenesis and under stressful conditions and contribute to further study on enhancing stress tolerance and disease resistance of mandarin fish.

  11. Interspecies Somatic Cell Nuclear Transfer: Advancements and Problems

    PubMed Central

    Lagutina, Irina; Fulka, Helena; Lazzari, Giovanna

    2013-01-01

    Abstract Embryologists working with livestock species were the pioneers in the field of reprogramming by somatic cell nuclear transfer (SCNT). Without the “Dolly experiment,” the field of cellular reprogramming would have been slow and induced plutipotent cells (iPSCs) would not have been conceived. The major drive of the work in mammalian cloning was the interest of the breeding industry to propagate superior genotypes. Soon it was realized that the properties of oocytes could be used also to clone endangered mammalian species or to reprogram the genomes of unrelated species through what is known as interspecies (i) SCNT, using easily available oocytes of livestock species. iSCNT for cloning animals works only for species that can interbreed, and experiments with taxonomically distant species have not been successful in obtaining live births or deriving embryonic stem cell (ESC) lines to be used for regenerative medicine. There are controversial reports in the literature, but in most cases these experiments have underlined some of the cellular and molecular mechanisms that are incomplete during cell nucleus reprogramming, including the failure to organize nucleoli, silence somatic cell genes, activate the embryonic genome, and resume mitochondrial replication and function, thus indicating nucleus–cytoplasmic incompatibility. PMID:24033141

  12. Somatic expression of LINE-1 elements in human tissues.

    PubMed

    Belancio, Victoria P; Roy-Engel, Astrid M; Pochampally, Radhika R; Deininger, Prescott

    2010-07-01

    LINE-1 expression damages host DNA via insertions and endonuclease-dependent DNA double-strand breaks (DSBs) that are highly toxic and mutagenic. The predominant tissue of LINE-1 expression has been considered to be the germ line. We show that both full-length and processed L1 transcripts are widespread in human somatic tissues and transformed cells, with significant variation in both L1 expression and L1 mRNA processing. This is the first demonstration that RNA processing is a major regulator of L1 activity. Many tissues also produce translatable spliced transcript (SpORF2). An Alu retrotransposition assay, COMET assays and 53BP1 foci staining show that the SpORF2 product can support functional ORF2 protein expression and can induce DNA damage in normal cells. Tests of the senescence-associated beta-galactosidase expression suggest that expression of exogenous full-length L1, or the SpORF2 mRNA alone in human fibroblasts and adult stem cells triggers a senescence-like phenotype, which is one of the reported responses to DNA damage. In contrast to previous assumptions that L1 expression is germ line specific, the increased spectrum of tissues exposed to L1-associated damage suggests a role for L1 as an endogenous mutagen in somatic tissues. These findings have potential consequences for the whole organism in the form of cancer and mammalian aging.

  13. Vitamin C modulates TET1 function during somatic cell reprogramming.

    PubMed

    Chen, Jiekai; Guo, Lin; Zhang, Lei; Wu, Haoyu; Yang, Jiaqi; Liu, He; Wang, Xiaoshan; Hu, Xiao; Gu, Tianpeng; Zhou, Zhiwei; Liu, Jing; Liu, Jiadong; Wu, Hongling; Mao, Shi-Qing; Mo, Kunlun; Li, Yingying; Lai, Keyu; Qi, Jing; Yao, Hongjie; Pan, Guangjin; Xu, Guo-Liang; Pei, Duanqing

    2013-12-01

    Vitamin C, a micronutrient known for its anti-scurvy activity in humans, promotes the generation of induced pluripotent stem cells (iPSCs) through the activity of histone demethylating dioxygenases. TET hydroxylases are also dioxygenases implicated in active DNA demethylation. Here we report that TET1 either positively or negatively regulates somatic cell reprogramming depending on the absence or presence of vitamin C. TET1 deficiency enhances reprogramming, and its overexpression impairs reprogramming in the context of vitamin C by modulating the obligatory mesenchymal-to-epithelial transition (MET). In the absence of vitamin C, TET1 promotes somatic cell reprogramming independent of MET. Consistently, TET1 regulates 5-hydroxymethylcytosine (5hmC) formation at loci critical for MET in a vitamin C-dependent fashion. Our findings suggest that vitamin C has a vital role in determining the biological outcome of TET1 function at the cellular level. Given its benefit to human health, vitamin C should be investigated further for its role in epigenetic regulation. PMID:24162740

  14. Metaplasia and somatic cell reprogramming.

    PubMed

    Slack, J M W

    2009-01-01

    The nature and occurrence of metaplasia is briefly reviewed. A theory of how metaplasia is initiated is presented, depending on the idea that it represents an alteration in the combination of developmental transcription factors that are expressed. Two examples of experimental metaplasia, provoked by over-expression of specific transcription factors, are presented: the transformation of B lymphocytes to macrophages, and of pancreatic exocrine cells to hepatocytes. The formation of induced pluripotential stem cells (iPS cells) is considered an example of the same process, in which the destination state is the embryonic stem cell. It is noted that iPS cell production is a stochastic process, depending on selection to obtain the desired cell type. It is proposed that analogous technology, using the appropriate transcription factors, could be used to bring about transformation to cell types other than embryonic stem cells. PMID:18855879

  15. Acetylcholinesterase-Inhibition and Antibacterial Activity of Mondia whitei Adventitious Roots and Ex vitro-Grown Somatic Embryogenic-Biomass

    PubMed Central

    Baskaran, Ponnusamy; Kumari, Aloka; Ncube, Bhekumthetho; Van Staden, Johannes

    2016-01-01

    Mondia whitei (Hook.f.) Skeels is an important endangered medicinal and commercial plant in South Africa. In vitro propagation systems are required for biomass production and bioactivity analysis to supplement wild resources/stocks. Adventitious roots from somatic embryogenic explants using suspension culture and ex vitro-grown plants produced via somatic embryogenesis were established using different plant growth regulator treatments. The adventitious root biomass and different parts of ex vitro-grown and mother plants were used to investigate the potential for acetylcholinesterase (AChE) and antibacterial activities. Adventitious roots derived from 2.5 μM indole-3-acetic acid (IAA) treatments and ex vitro-grown plants derived from meta-topolin riboside and IAA treatments gave the best AChE and antibacterial activities. The in vitro-established M. whitei and ex vitro biomass have comparable ability to function as inhibitors of acetylcholinesterase and antibacterial agents, and can be used as potent bioresources in traditional medicine. PMID:27752244

  16. Bone morphogenetic protein 15 (BMP15) acts as a BMP and Wnt inhibitor during early embryogenesis.

    PubMed

    Di Pasquale, Elisa; Brivanlou, Ali H

    2009-09-18

    Bone morphogenetic protein 15 (BMP15) belongs to an unusual subgroup of the transforming growth factor beta (TGFbeta) 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 beta-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.

  17. Effects of chronic administration of delta-9-tetrahydrocannabinol on early embryogenesis of the mouse.

    PubMed

    Shinohara, O; Henrich, R T; Morishima, A

    1983-10-01

    The effects of chronic administration of delta-9-tetrahydrocannabinol (THC), the major psychoactive component of marihuana, on embryogenesis were investigated in the mouse treated with THC prior to sexual maturation. The study was designed to imitate the prolonged abuse of marihuana by sexually immature and adolescent girls. Female Swiss-Webster mice were injected, intraperitoneally, with THC 5 mg/kg per day or with the vehicle for 21 consecutive days, starting on the 30th day of life, which is prior to their sexual maturation. The dose of THC was approximately equivalent to that absorbed by a man smoking 1 to 2 marihuana cigarettes per day. Superovulation was then induced, and the mice were mated with untreated males. Ova were recovered from the oviducts just prior to the first cleavage division, and at about the time of the second cleavage division. The incidence of degenerative ova increased from 13.6% in the controls to 19.1% in the THC-treated group (P less than 0.001) at the time of the second cleavage division, but there was no difference in the incidence of morphologically abnormal ova between the two groups just prior to the first cleavage division. The rate of successful completion of the first cleavage division, but not the second cleavage division, was adversely affected by THC, resulting in accumulation of morphologically abnormal ova at the later stage. In view of the known disruptive effect of THC on the process of cell division, it was postulated that THC might have adversely affected meiosis, leading to the inability of the ova to undergo the first cleavage division.

  18. LEA (Late Embryogenesis Abundant) proteins and their encoding genes in Arabidopsis thaliana

    PubMed Central

    Hundertmark, Michaela; Hincha, Dirk K

    2008-01-01

    Background LEA (late embryogenesis abundant) proteins have first been described about 25 years ago as accumulating late in plant seed development. They were later found in vegetative plant tissues following environmental stress and also in desiccation tolerant bacteria and invertebrates. Although they are widely assumed to play crucial roles in cellular dehydration tolerance, their physiological and biochemical functions are largely unknown. Results We present a genome-wide analysis of LEA proteins and their encoding genes in Arabidopsis thaliana. We identified 51 LEA protein encoding genes in the Arabidopsis genome that could be classified into nine distinct groups. Expression studies were performed on all genes at different developmental stages, in different plant organs and under different stress and hormone treatments using quantitative RT-PCR. We found evidence of expression for all 51 genes. There was only little overlap between genes expressed in vegetative tissues and in seeds and expression levels were generally higher in seeds. Most genes encoding LEA proteins had abscisic acid response (ABRE) and/or low temperature response (LTRE) elements in their promoters and many genes containing the respective promoter elements were induced by abscisic acid, cold or drought. We also found that 33% of all Arabidopsis LEA protein encoding genes are arranged in tandem repeats and that 43% are part of homeologous pairs. The majority of LEA proteins were predicted to be highly hydrophilic and natively unstructured, but some were predicted to be folded. Conclusion The analyses indicate a wide range of sequence diversity, intracellular localizations, and expression patterns. The high fraction of retained duplicate genes and the inferred functional diversification indicate that they confer an evolutionary advantage for an organism under varying stressful environmental conditions. This comprehensive analysis will be an important starting point for future efforts to elucidate

  19. Zebrafish Noxa promotes mitosis in early embryonic development and regulates apoptosis in subsequent embryogenesis.

    PubMed

    Zhong, J-X; Zhou, L; Li, Z; Wang, Y; Gui, J-F

    2014-06-01

    Noxa functions in apoptosis and immune system of vertebrates, but its activities in embryo development remain unclear. In this study, we have studied the role of zebrafish Noxa (zNoxa) by using zNoxa-specifc morpholino knockdown and overexpression approaches in developing zebrafish embryos. Expression pattern analysis indicates that zNoxa transcript is of maternal origin, which displays a uniform distribution in early embryonic development until shield stage, and the zygote zNoxa transcription is initiated from this stage and mainly localized in YSL of the embryos. The zNoxa expression alterations result in strong embryonic development defects, demonstrating that zNoxa regulates apoptosis from 75% epiboly stage of development onward, in which zNoxa firstly induces the expression of zBik, and then cooperates with zBik to regulate apoptosis. Moreover, zNoxa knockdown also causes a reduction in number of mitotic cells before 8 h.p.f., suggesting that zNoxa also promotes mitosis before 75% epiboly stage. The effect of zNoxa on mitosis is mediated by zWnt4b in early embryos, whereas zMcl1a and zMcl1b suppress the ability of zNoxa to regulate mitosis and apoptosis at different developmental stages. In addition, mammalian mouse Noxa (mNoxa) mRNA was demonstrated to rescue the arrest of mitosis when zNoxa was knocked down, suggesting that mouse and zebrafish Noxa might have similar dual functions. Therefore, the current findings indicate that Noxa is a novel regulator of early mitosis before 75% epiboly stage when it translates into a key mediator of apoptosis in subsequent embryogenesis.

  20. Tissue culture-induced genetic and epigenetic variation in triticale (× Triticosecale spp. Wittmack ex A. Camus 1927) regenerants.

    PubMed

    Machczyńska, Joanna; Zimny, Janusz; Bednarek, Piotr Tomasz

    2015-10-01

    Plant regeneration via in vitro culture can induce genetic and epigenetic variation; however, the extent of such changes in triticale is not yet understood. In the present study, metAFLP, a variation of methylation-sensitive amplified fragment length polymorphism analysis, was used to investigate tissue culture-induced variation in triticale regenerants derived from four distinct genotypes using androgenesis and somatic embryogenesis. The metAFLP technique enabled identification of both sequence and DNA methylation pattern changes in a single experiment. Moreover, it was possible to quantify subtle effects such as sequence variation, demethylation, and de novo methylation, which affected 19, 5.5, 4.5% of sites, respectively. Comparison of variation in different genotypes and with different in vitro regeneration approaches demonstrated that both the culture technique and genetic background of donor plants affected tissue culture-induced variation. The results showed that the metAFLP approach could be used for quantification of tissue culture-induced variation and provided direct evidence that in vitro plant regeneration could cause genetic and epigenetic variation.

  1. Tissue culture-induced genetic and epigenetic variation in triticale (× Triticosecale spp. Wittmack ex A. Camus 1927) regenerants.

    PubMed

    Machczyńska, Joanna; Zimny, Janusz; Bednarek, Piotr Tomasz

    2015-10-01

    Plant regeneration via in vitro culture can induce genetic and epigenetic variation; however, the extent of such changes in triticale is not yet understood. In the present study, metAFLP, a variation of methylation-sensitive amplified fragment length polymorphism analysis, was used to investigate tissue culture-induced variation in triticale regenerants derived from four distinct genotypes using androgenesis and somatic embryogenesis. The metAFLP technique enabled identification of both sequence and DNA methylation pattern changes in a single experiment. Moreover, it was possible to quantify subtle effects such as sequence variation, demethylation, and de novo methylation, which affected 19, 5.5, 4.5% of sites, respectively. Comparison of variation in different genotypes and with different in vitro regeneration approaches demonstrated that both the culture technique and genetic background of donor plants affected tissue culture-induced variation. The results showed that the metAFLP approach could be used for quantification of tissue culture-induced variation and provided direct evidence that in vitro plant regeneration could cause genetic and epigenetic variation. PMID:26337939

  2. 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

  3. Emerging patterns of somatic mutations in cancer.

    PubMed

    Watson, Ian R; Takahashi, Koichi; Futreal, P Andrew; Chin, Lynda

    2013-10-01

    Recent advances in technological tools for massively parallel, high-throughput sequencing of DNA have enabled the comprehensive characterization of somatic mutations in a large number of tumour samples. In this Review, we describe 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 and spectra, as well as the roles of environmental insults that influence these processes. We highlight the developing statistical approaches that are used to identify significantly mutated genes, and discuss the emerging biological and clinical insights from such analyses, as well as the future challenges of translating these genomic data into clinical impacts.

  4. Glucose 6-phosphate dehydrogenase deficiency enhances germ cell apoptosis and causes defective embryogenesis in Caenorhabditis elegans

    PubMed Central

    Yang, H-C; Chen, T-L; Wu, Y-H; Cheng, K-P; Lin, Y-H; Cheng, M-L; Ho, H-Y; Lo, S J; Chiu, D T-Y

    2013-01-01

    Glucose 6-phosphate dehydrogenase (G6PD) deficiency, known as favism, is classically manifested by hemolytic anemia in human. More recently, it has been shown that mild G6PD deficiency moderately affects cardiac function, whereas severe G6PD deficiency leads to embryonic lethality in mice. How G6PD deficiency affects organisms has not been fully elucidated due to the lack of a suitable animal model. In this study, G6PD-deficient Caenorhabditis elegans was established by RNA interference (RNAi) knockdown to delineate the role of G6PD in animal physiology. Upon G6PD RNAi knockdown, G6PD activity was significantly hampered in C. elegans in parallel with increased oxidative stress and DNA oxidative damage. Phenotypically, G6PD-knockdown enhanced germ cell apoptosis (2-fold increase), reduced egg production (65% of mock), and hatching (10% of mock). To determine whether oxidative stress is associated with G6PD knockdown-induced reproduction defects, C. elegans was challenged with a short-term hydrogen peroxide (H2O2). The early phase egg production of both mock and G6PD-knockdown C. elegans were significantly affected by H2O2. However, H2O2-induced germ cell apoptosis was more dramatic in mock than that in G6PD-deficient C. elegans. To investigate the signaling pathways involved in defective oogenesis and embryogenesis caused by G6PD knockdown, mutants of p53 and mitogen-activated protein kinase (MAPK) pathways were examined. Despite the upregulation of CEP-1 (p53), cep-1 mutation did not affect egg production and hatching in G6PD-deficient C. elegans. Neither pmk-1 nor mek-1 mutation significantly affected egg production, whereas sek-1 mutation further decreased egg production in G6PD-deficient C. elegans. Intriguingly, loss of function of sek-1 or mek-1 dramatically rescued defective hatching (8.3- and 9.6-fold increase, respectively) induced by G6PD knockdown. Taken together, these findings show that G6PD knockdown reduces egg production and hatching in C. elegans

  5. Handmade somatic cell cloning in cattle.

    PubMed

    Vajta, Gàbor; Lewis, Ian M; Tecirlioglu, R Tayfur

    2006-01-01

    Apart from the biological and ethical problems, technical difficulties also hamper the improvement and widespread application of somatic cell nuclear transfer (NT). Recently introduced zona-free procedures may offer a solution for the latter problem. The most radical approach of these techniques is the so-called handmade cloning (HMC). It does not require micromanipulators because the manipulations required for both enucleation and nucleus transfer are performed by hand. The HMC technique includes manual bisection of zona-free oocytes, selection of cytoplasts by staining, and the simultaneous fusion of the somatic cell with two cytoplasts to produce a cloned embryo. HMC is a rapid and efficient technique that suits large-scale NT programs. It requires less expertise and time than traditional NT methods and the cost of equipment is significantly less. Production efficiency is high and embryo quality, in terms of pregnancy rates and live births, is not compromised. Although HMC has been developed particularly for bovine NT, the technique is applicable to other species. The method may become a useful tool for both experimental and commercial somatic cell cloning because it allows for standardization of procedures and provides the possibility of automation.

  6. 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.

  7. The social evolution of somatic fusion.

    PubMed

    Aanen, Duur K; Debets, Alfons J M; de Visser, J Arjan G M; Hoekstra, Rolf F

    2008-11-01

    The widespread potential for somatic fusion among different conspecific multicellular individuals suggests that such fusion is adaptive. However, because recognition of non-kin (allorecognition) usually leads to a rejection response, successful somatic fusion is limited to close kin. This is consistent with kin-selection theory, which predicts that the potential cost of fusion and the potential for somatic parasitism decrease with increasing relatedness. Paradoxically, however, Crozier found that, in the short term, positive-frequency-dependent selection eliminates the required genetic polymorphism at allorecognition loci. The 'Crozier paradox' may be solved if allorecognition is based on extrinsically balanced polymorphisms, for example at immune loci. Alternatively, the assumption of most models that self fusion is mutually beneficial is wrong. If fusion is on average harmful, selection will promote unconditional rejection. However, we propose that fusion within individuals is beneficial, selecting for the ability to fuse, but fusion between individuals on average costly, selecting for non-self recognition (rather than non-kin recognition). We discuss experimental data on fungi that are consistent with this hypothesis. PMID:18937373

  8. Cloned mice derived from somatic cell nuclei.

    PubMed

    Hosaka, K; Ohi, S; Ando, A; Kobayashi, M; Sato, K

    2000-12-01

    In 1997, a cloned sheep "Dolly" was produced by nuclear transfer of somatic cell. The first birth of cloned mice derived from some somatic cells were succeeded in 1998. At present, it is shown that somatic cells, cumulus cells, fibroblasts and Sertoli cells can be used to the study of cloned animal as nuclear donor. In this study investigation was designed to compare with efficiency on the production of cloned embryos by using the microinjection and the electrofusion methods for nuclear transfer. Oocyte enucleation was performed with a micromanipulator. The oocyte was held by holding pipette, and was enucleated using a beveled pipette. Microinjection method: Cell's nucleus injection was carried out by piezo-micromanipulator. Cytochalasin B treated cumulus cell was aspirated into a injection pipette, and was broken its plasma membrane using the injection pipette. Then, the cumulus cell was injected into the enucleated ooplasm directly. Electrofusion method: The cell was aspirated into a beveled pipette, and then an aspirated cell was inserted into perivitelline space. Then, the pair of enucleated oocyte and cell was fused using electrical cell fusion apparatus. The reconstituted embryos were activated after nuclear transfer using St2+. Reconstituted embryos had been produced by the microinjection showed the embryonic development to over 8-cell stages. But, the rate of fragmentation of reconstituted embryos by the microinjection showed a little high rate in comparison with the electrofusion. When some reconstituted embryos by the microinjection were transplanted to pseudopregnant females' oviduct, 9 fetuses were observed at 14 days post coitum. PMID:11329940

  9. Glycogen Synthase Kinase-3 is involved in glycogen metabolism control and embryogenesis of Rhodnius prolixus.

    PubMed

    Mury, Flávia B; Lugon, Magda D; DA Fonseca, Rodrigo Nunes; Silva, Jose R; Berni, Mateus; Araujo, Helena M; Fontenele, Marcio Ribeiro; Abreu, Leonardo Araujo DE; Dansa, Marílvia; Braz, Glória; Masuda, Hatisaburo; Logullo, Carlos

    2016-10-01

    Rhodnius prolixus is a blood-feeding insect that transmits Trypanosoma cruzi and Trypanosoma rangeli to vertebrate hosts. Rhodnius prolixus is also a classical model in insect physiology, and the recent availability of R. prolixus genome has opened new avenues on triatomine research. Glycogen synthase kinase 3 (GSK-3) is classically described as a key enzyme involved in glycogen metabolism, also acting as a downstream component of the Wnt pathway during embryogenesis. GSK-3 has been shown to be highly conserved among several organisms, mainly in the catalytic domain region. Meanwhile, the role of GSK-3 during R. prolixus embryogenesis or glycogen metabolism has not been investigated. Here we show that chemical inhibition of GSK-3 by alsterpaullone, an ATP-competitive inhibitor of GSK3, does not affect adult survival rate, though it alters oviposition and egg hatching. Specific GSK-3 gene silencing by dsRNA injection in adult females showed a similar phenotype. Furthermore, bright field and 4'-6-diamidino-2-phenylindole (DAPI) staining analysis revealed that ovaries and eggs from dsGSK-3 injected females exhibited specific morphological defects. We also demonstrate that glycogen content was inversely related to activity and transcription levels of GSK-3 during embryogenesis. Lastly, after GSK-3 knockdown, we observed changes in the expression of the Wingless (Wnt) downstream target β-catenin as well as in members of other pathways such as the receptor Notch. Taken together, our results show that GSK-3 regulation is essential for R. prolixus oogenesis and embryogenesis.

  10. Glycogen Synthase Kinase-3 is involved in glycogen metabolism control and embryogenesis of Rhodnius prolixus.

    PubMed

    Mury, Flávia B; Lugon, Magda D; DA Fonseca, Rodrigo Nunes; Silva, Jose R; Berni, Mateus; Araujo, Helena M; Fontenele, Marcio Ribeiro; Abreu, Leonardo Araujo DE; Dansa, Marílvia; Braz, Glória; Masuda, Hatisaburo; Logullo, Carlos

    2016-10-01

    Rhodnius prolixus is a blood-feeding insect that transmits Trypanosoma cruzi and Trypanosoma rangeli to vertebrate hosts. Rhodnius prolixus is also a classical model in insect physiology, and the recent availability of R. prolixus genome has opened new avenues on triatomine research. Glycogen synthase kinase 3 (GSK-3) is classically described as a key enzyme involved in glycogen metabolism, also acting as a downstream component of the Wnt pathway during embryogenesis. GSK-3 has been shown to be highly conserved among several organisms, mainly in the catalytic domain region. Meanwhile, the role of GSK-3 during R. prolixus embryogenesis or glycogen metabolism has not been investigated. Here we show that chemical inhibition of GSK-3 by alsterpaullone, an ATP-competitive inhibitor of GSK3, does not affect adult survival rate, though it alters oviposition and egg hatching. Specific GSK-3 gene silencing by dsRNA injection in adult females showed a similar phenotype. Furthermore, bright field and 4'-6-diamidino-2-phenylindole (DAPI) staining analysis revealed that ovaries and eggs from dsGSK-3 injected females exhibited specific morphological defects. We also demonstrate that glycogen content was inversely related to activity and transcription levels of GSK-3 during embryogenesis. Lastly, after GSK-3 knockdown, we observed changes in the expression of the Wingless (Wnt) downstream target β-catenin as well as in members of other pathways such as the receptor Notch. Taken together, our results show that GSK-3 regulation is essential for R. prolixus oogenesis and embryogenesis. PMID:27574112

  11. 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…

  12. In-depth proteomics characterization of embryogenesis of the honey bee worker (Apis mellifera ligustica).

    PubMed

    Fang, Yu; Feng, Mao; Han, Bin; Lu, Xiaoshan; Ramadan, Haitham; Li, Jianke

    2014-09-01

    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

  13. In-depth proteomics characterization of embryogenesis of the honey bee worker (Apis mellifera ligustica).

    PubMed

    Fang, Yu; Feng, Mao; Han, Bin; Lu, Xiaoshan; Ramadan, Haitham; Li, Jianke

    2014-09-01

    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

  14. 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

  15. Germ band retraction as a landmark in glucose metabolism during Aedes aegypti embryogenesis

    PubMed Central

    2010-01-01

    Background The mosquito A. aegypti is vector of dengue and other viruses. New methods of vector control are needed and can be achieved by a better understanding of the life cycle of this insect. Embryogenesis is a part of A. aegypty life cycle that is poorly understood. In insects in general and in mosquitoes in particular energetic metabolism is well studied during oogenesis, when the oocyte exhibits fast growth, accumulating carbohydrates, lipids and proteins that will meet the regulatory and metabolic needs of the developing embryo. On the other hand, events related with energetic metabolism during A. aegypti embryogenesis are unknown. Results Glucose metabolism was investigated throughout Aedes aegypti (Diptera) embryonic development. Both cellular blastoderm formation (CBf, 5 h after egg laying - HAE) and germ band retraction (GBr, 24 HAE) may be considered landmarks regarding glucose 6-phosphate (G6P) destination. We observed high levels of glucose 6-phosphate dehydrogenase (G6PDH) activity at the very beginning of embryogenesis, which nevertheless decreased up to 5 HAE. This activity is correlated with the need for nucleotide precursors generated by the pentose phosphate pathway (PPP), of which G6PDH is the key enzyme. We suggest the synchronism of egg metabolism with carbohydrate distribution based on the decreasing levels of phosphoenolpyruvate carboxykinase (PEPCK) activity and on the elevation observed in protein content up to 24 HAE. Concomitantly, increasing levels of hexokinase (HK) and pyruvate kinase (PK) activity were observed, and PEPCK reached a peak around 48 HAE. Glycogen synthase kinase (GSK3) activity was also monitored and shown to be inversely correlated with glycogen distribution during embryogenesis. Conclusions The results herein support the hypothesis that glucose metabolic fate changes according to developmental embryonic stages. Germ band retraction is a moment that was characterized as a landmark in glucose metabolism during Aedes

  16. Thyroid hormone receptor can modulate retinoic acid-mediated axis formation in frog embryogenesis.

    PubMed Central

    Banker, D E; Eisenman, R N

    1993-01-01

    Thyroid hormone receptor acts as a hormone-dependent transcriptional transactivator and as a transcriptional repressor in the absence of thyroid hormone. Specifically, thyroid hormone receptor can repress retinoic acid-induced gene expression through interactions with retinoic acid receptor. (Retinoic acid is a potent teratogen in the frog Xenopus laevis, acting at early embryonic stages to interfere with the formation of anterior structures. Endogenous retinoic acid is thought to act in normal anterior-posterior axis formation.) We have previously shown that thyroid hormone receptor RNA (alpha isotype) is expressed and polysome-associated during Xenopus embryogenesis preceding thyroid gland maturation and endogenous thyroid hormone production (D. E. Banker, J. Bigler, and R. N. Eisenman, Mol. Cell. Biol. 11:5079-5089, 1991). To determine whether thyroid hormone receptor might influence the effects of retinoic acid in early frog development, we have examined the results of ectopic thyroid hormone receptor expression on retinoic acid teratogenesis. We demonstrate that microinjections of full-length thyroid hormone receptor RNA protect injected embryos from retinoic acid teratogenesis. DNA binding is apparently essential to this protective function, as truncated thyroid hormone receptors, lacking DNA-binding domains but including hormone-binding and dimerization domains, do not protect from retinoic acid. We have shown that microinjections of these dominant-interfering thyroid hormone receptors, as well as anti-thyroid hormone receptor antibodies, increase retinoic acid teratogenesis in injected embryos, presumably by inactivating endogenous thyroid hormone receptor. This finding suggests that endogenous thyroid hormone receptors may act to limit retinoic acid sensitivity. On the other hand, after thyroid hormone treatment, ectopic thyroid hormone receptor mediates teratogenesis that is indistinguishable from the dorsoanterior deficiencies produced in retinoic acid

  17. [Functional somatic pain syndromes: summary of hypotheses of their overlap and etiology].

    PubMed

    Henningsen, P; Derra, C; Türp, J C; Häuser, W

    2004-04-01

    Currently it is unclear whether functional somatic syndromes can be explained by one common underlying functional syndrome. In any case it does not seem justified to view functional somatic syndromes as purely psychological disorders (somatized anxiety or depression). Psychiatric comorbidity and life time stress including traumatisations are mainly, but not exclusively responsible for triggering health care utilisation. The lowered pain threshold that can be demonstrated clinically and experimentally in fibromyalgia, irritable bowel syndrome, tension headache and temporomandibular disorders is currently seen primarily as result of an altered central nervous processing of nociceptive input. In addition some results also hint at a disturbance in the hypothalamus-pituitary-adrenal axis. The predominance of female patients can be due to gender specific illness behaviour as well as to estrogen-induced changes in pain sensitivity. In sum, functional somatic syndromes currently are best explained by a biopsychosocial model of predisposing, triggering and maintaining factors. More research is needed particularly to clarify the role of genetic and of cultural factors. PMID:15067534

  18. Somatosensory Pulsatile Tinnitus Syndrome: Somatic Testing Identifies a Pulsatile Tinnitus Subtype That Implicates the Somatosensory System

    PubMed Central

    Levine, Robert Aaron; Nam, Eui-Cheol; Melcher, Jennifer

    2008-01-01

    A new tinnitus syndrome is described: high-pitched, cardiac-synchronous tinnitus, whose pulsations are suppressed by strong contractions or compressions of the neck and jaw muscles (somatic testing). 14 cases, 6 non-lateralized and 8 unilateral, are reported. In the non-lateralized cases, onset was bilateral. In the one intermittent case, while her tinnitus was absent her pulsatile tinnitus could be induced by somatic testing. No etiology was found from physical examination, imaging, or ancillary testing. Because these cases of pulsatile tinnitus can be both induced and suppressed by activation of the somatosensory system of the head or upper lateral neck, we propose that this syndrome is occurring from (a) cardiac synchronous somatosensory activation of the central auditory pathway or (b) failure of the somatosensory-auditory central nervous system interactions to suppress cardiac somatosounds. PMID:18632767

  19. A XEN-like State Bridges Somatic Cells to Pluripotency during Chemical Reprogramming.

    PubMed

    Zhao, Yang; Zhao, Ting; Guan, Jingyang; Zhang, Xu; Fu, Yao; Ye, Junqing; Zhu, Jialiang; Meng, Gaofan; Ge, Jian; Yang, Susu; Cheng, Lin; Du, Yaqin; Zhao, Chaoran; Wang, Ting; Su, Linlin; Yang, Weifeng; Deng, Hongkui

    2015-12-17

    Somatic cells can be reprogrammed into pluripotent stem cells (PSCs) by using pure chemicals, providing a different paradigm to study somatic reprogramming. However, the cell fate dynamics and molecular events that occur during the chemical reprogramming process remain unclear. We now show that the chemical reprogramming process requires the early formation of extra-embryonic endoderm (XEN)-like cells and a late transition from XEN-like cells to chemically-induced (Ci)PSCs, a unique route that fundamentally differs from the pathway of transcription factor-induced reprogramming. Moreover, precise manipulation of the cell fate transition in a step-wise manner through the XEN-like state allows us to identify small-molecule boosters and establish a robust chemical reprogramming system with a yield up to 1,000-fold greater than that of the previously reported protocol. These findings demonstrate that chemical reprogramming is a promising approach to manipulate cell fates.

  20. Transient acquisition of pluripotency during somatic cell transdifferentiation with iPSC reprogramming factors.

    PubMed

    Maza, Itay; Caspi, Inbal; Zviran, Asaf; Chomsky, Elad; Rais, Yoach; Viukov, Sergey; Geula, Shay; Buenrostro, Jason D; Weinberger, Leehee; Krupalnik, Vladislav; Hanna, Suhair; Zerbib, Mirie; Dutton, James R; Greenleaf, William J; Massarwa, Rada; Novershtern, Noa; Hanna, Jacob H

    2015-07-01

    Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced 'transdifferentiation' pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by various methods.

  1. amontillado, the Drosophila homolog of the prohormone processing protease PC2, is required during embryogenesis and early larval development.

    PubMed Central

    Rayburn, Lowell Y M; Gooding, Holly C; Choksi, Semil P; Maloney, Dhea; Kidd, Ambrose R; Siekhaus, Daria E; Bender, Michael

    2003-01-01

    Biosynthesis of most peptide hormones and neuropeptides requires proteolytic excision of the active peptide from inactive proprotein precursors, an activity carried out by subtilisin-like proprotein convertases (SPCs) in constitutive or regulated secretory pathways. The Drosophila amontillado (amon) gene encodes a homolog of the mammalian PC2 protein, an SPC that functions in the regulated secretory pathway in neuroendocrine tissues. We have identified amon mutants by isolating ethylmethanesulfonate (EMS)-induced lethal and visible mutations that define two complementation groups in the amon interval at 97D1 of the third chromosome. DNA sequencing identified the amon complementation group and the DNA sequence change for each of the nine amon alleles isolated. amon mutants display partial embryonic lethality, are defective in larval growth, and arrest during the first to second instar larval molt. Mutant larvae can be rescued by heat-shock-induced expression of the amon protein. Rescued larvae arrest at the subsequent larval molt, suggesting that amon is also required for the second to third instar larval molt. Our data indicate that the amon proprotein convertase is required during embryogenesis and larval development in Drosophila and support the hypothesis that AMON acts to proteolytically process peptide hormones that regulate hatching, larval growth, and larval ecdysis. PMID:12586710

  2. Induced Pluripotent Stem Cells and Their Use in Cardiac and Neural Regenerative Medicine

    PubMed Central

    Skalova, Stepanka; Svadlakova, Tereza; Qureshi, Wasay Mohiuddin Shaikh; Dev, Kapil; Mokry, Jaroslav

    2015-01-01

    Stem cells are unique pools of cells that are crucial for embryonic development and maintenance of adult tissue homeostasis. The landmark Nobel Prize winning research by Yamanaka and colleagues to induce pluripotency in somatic cells has reshaped the field of stem cell research. The complications related to the usage of pluripotent embryonic stem cells (ESCs) in human medicine, particularly ESC isolation and histoincompatibility were bypassed with induced pluripotent stem cell (iPSC) technology. The human iPSCs can be used for studying embryogenesis, disease modeling, drug testing and regenerative medicine. iPSCs can be diverted to different cell lineages using small molecules and growth factors. In this review we have focused on iPSC differentiation towards cardiac and neuronal lineages. Moreover, we deal with the use of iPSCs in regenerative medicine and modeling diseases like myocardial infarction, Timothy syndrome, dilated cardiomyopathy, Parkinson’s, Alzheimer’s and Huntington’s disease. Despite the promising potential of iPSCs, genome contamination and low efficacy of cell reprogramming remain significant challenges. PMID:25689424

  3. A SOMATIC-MARKER THEORY OF ADDICTION

    PubMed Central

    Verdejo-García, Antonio; Bechara, Antoine

    2009-01-01

    Similar to patients with ventromedial prefrontal cortex (VMPC) lesions, substance abusers show altered decision-making, characterized by a tendency to choose the immediate reward, at the expense of negative future consequences. The somatic-marker model proposes that decision-making depends on neural substrates that regulate homeostasis, emotion and feeling. According to this model, there should be a link between alterations in processing emotions in substance abusers, and their impairments in decision-making. A growing evidence from neuroscientific studies indicate that core aspects of addiction may be explained in terms of abnormal emotional/homeostatic guidance of decision-making. Behavioural studies have revealed emotional processing and decision-making deficits in substance abusers. Neuroimaging studies have shown that altered decision-making in addiction is associated with abnormal functioning of a distributed neural network critical for the processing of emotional information, and the experience of “craving”, including the VMPC, the amygdala, the striatum, the anterior cingulate cortex, and the insular/somato-sensory cortices, as well as non-specific neurotransmitter systems that modulate activities of neural processes involved in decision-making. The aim of this paper is to review this growing evidence, and to examine the extent of which these studies support a somatic-marker theory of addiction. We conclude that there are at least two underlying types of dysfunctions where emotional signals (somatic-markers) turns in favor of immediate outcomes in addiction: (1) a hyperactivity in the amygdala or impulsive system, which exaggerates the rewarding impact of available incentives, and (2) hypoactivity in the prefrontal cortex or reflective system, which forecasts the long-term consequences of a given action. PMID:18722390

  4. 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

  5. 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

  6. 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.

  7. [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

  8. Confidence-based somatic mutation evaluation and prioritization.

    PubMed

    Löwer, Martin; Renard, Bernhard Y; de Graaf, Jos; Wagner, Meike; Paret, Claudia; Kneip, Christoph; Türeci, Ozlem; Diken, Mustafa; Britten, Cedrik; Kreiter, Sebastian; Koslowski, Michael; Castle, John C; Sahin, Ugur

    2012-01-01

    Next generation sequencing (NGS) has enabled high throughput discovery of somatic mutations. Detection depends on experimental design, lab platforms, parameters and analysis algorithms. However, NGS-based somatic mutation detection is prone to erroneous calls, with reported validation rates near 54% and congruence between algorithms less than 50%. Here, we developed an algorithm to assign a single statistic, a false discovery rate (FDR), to each somatic mutation identified by NGS. This FDR confidence value accurately discriminates true mutations from erroneous calls. Using sequencing data generated from triplicate exome profiling of C57BL/6 mice and B16-F10 melanoma cells, we used the existing algorithms GATK, SAMtools and SomaticSNiPer to identify somatic mutations. For each identified mutation, our algorithm assigned an FDR. We selected 139 mutations for validation, including 50 somatic mutations assigned a low FDR (high confidence) and 44 mutations assigned a high FDR (low confidence). All of the high confidence somatic mutations validated (50 of 50), none of the 44 low confidence somatic mutations validated, and 15 of 45 mutations with an intermediate FDR validated. Furthermore, the assignment of a single FDR to individual mutations enables statistical comparisons of lab and computation methodologies, including ROC curves and AUC metrics. Using the HiSeq 2000, single end 50 nt reads from replicates generate the highest confidence somatic mutation call set.

  9. Emotional distress in the Hebrew Bible. Somatic or psychological?

    PubMed

    Mumford, D B

    1992-01-01

    A systematic search was made in the Hebrew Bible for expressions of emotional distress. A wide range of somatic and psychological vocabulary was found, especially in the Psalms and other poetic literature. Somatic expressions most frequently involved the heart, bowels, belly, bones, and eyes. Head symptoms were rare. Metaphors referring to the heart were common; other somatic expressions appeared to be descriptions of actual physical sensations. Usually somatic and psychological expressions were paired together, utilising the 'parallelism' of Hebrew verse form. Biblical Hebrew thus incorporated a powerful and sophisticated language of emotional expression.

  10. 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

  11. Blastocysts derivation from somatic cell fusion with premature oocytes (prematuration somatic cell fusion).

    PubMed

    Saadeldin, Islam M; Khoirinaya, Candrani; Kim, Su Jin; Moon, Joon Ho; Almadaly, Essam; Lee, Byeong Chun

    2016-02-01

    This study was undertaken to investigate the development of immature oocytes after their fusion with male somatic cells expressing red fluorescence protein (RFP). RFP-expressing cells were fused with immature oocytes, matured in vitro and then parthenogenetically activated. Somatic nuclei showed spindle formation, 1st polar body extrusion after in vitro maturation and protruded the 2nd polar body after parthenogenetic activation. RFP was expressed in the resultant embryos; two-cell stage and blastocysts. Chromosomal analysis showed aneuploidy in 81.82% of the resulting blastocysts while 18.18% of the resulting blastocysts were diploid. Among eight RFP-expressing blastocysts, Xist mRNAs was detected in six while Sry mRNA was detected in only one blastocyst. We propose "prematuration somatic cell fusion" as an approach to generate embryos using somatic cells instead of spermatozoa. The current approach, if improved, would assist production of embryos for couples where the male partner is sterile, however, genetic and chromosomal analysis of the resultant embryos are required before transfer to the mothers.

  12. Cyclooxygenase and prostaglandins in somatic cell populations of the testis.

    PubMed

    Frungieri, Mónica B; Calandra, Ricardo S; Mayerhofer, Artur; Matzkin, María E

    2015-04-01

    Prostaglandins (PGs) are synthesized through the action of the rate-limiting enzyme cyclooxygenase (COX) and further specific enzymes. The development of Cox-deficient mice in the 1990s gave insights into the reproductive roles of PGs. Female Cox-knockout mice were subfertile or infertile. Interestingly, fertility was not affected in male mice deficient in Cox, suggesting that PGs may not be critical for the functioning of the testis. However, this conclusion has recently been challenged by observations of important roles for PGs in both physiological and pathological processes in the testis. The two key somatic cell types in the testis, Leydig and Sertoli cells, express the inducible isoenzyme COX2 and produce PGs. Testicular COX2 expression in these somatic cells is regulated by hormonal input (FSH, prolactin (PRL), and testosterone) as well as by IL1β. PGs modulate steroidogenesis in Leydig cells and glucose uptake in Sertoli cells. Hence, the COX2/PG system in Leydig and Sertoli cells acts as a local modulator of testicular activity, and consequently may regulate spermatogenic efficiency. In addition to its expression in Leydig and Sertoli cells, COX2 has been detected in the seminiferous tubule wall, and in testicular macrophages and mast cells of infertile patients. These observations highlight the possible relevance of PGs in testicular inflammation associated with idiopathic infertility. Collectively, these data indicate that the COX2/PG system plays crucial roles not only in testicular physiology (i.e., development, steroidogenesis, and spermatogenesis), but more importantly in the pathogenesis or maintenance of infertility status in the male gonad. Further studies of these actions could lead to new therapeutic approaches to idiopathic male infertility.

  13. Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells.

    PubMed

    Yamada, Mitsutoshi; Johannesson, Bjarki; Sagi, Ido; Burnett, Lisa Cole; Kort, Daniel H; Prosser, Robert W; Paull, Daniel; Nestor, Michael W; Freeby, Matthew; Greenberg, Ellen; Goland, Robin S; Leibel, Rudolph L; Solomon, Susan L; Benvenisty, Nissim; Sauer, Mark V; Egli, Dieter

    2014-06-26

    The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells that are consistently equivalent to embryonic stem cells, holding promise for autologous cell replacement therapy. Although methods to induce pluripotent stem cells from somatic cells by transcription factors are widely used in basic research, numerous differences between induced pluripotent stem cells and embryonic stem cells have been reported, potentially affecting their clinical use. Because of the therapeutic potential of diploid embryonic stem-cell lines derived from adult cells of diseased human subjects, we have systematically investigated the parameters affecting efficiency of blastocyst development and stem-cell derivation. Here we show that improvements to the oocyte activation protocol, including the use of both kinase and translation inhibitors, and cell culture in the presence of histone deacetylase inhibitors, promote development to the blastocyst stage. Developmental efficiency varied between oocyte donors, and was inversely related to the number of days of hormonal stimulation required for oocyte maturation, whereas the daily dose of gonadotropin or the total number of metaphase II oocytes retrieved did not affect developmental outcome. Because the use of concentrated Sendai virus for cell fusion induced an increase in intracellular calcium concentration, causing premature oocyte activation, we used diluted Sendai virus in calcium-free medium. Using this modified nuclear transfer protocol, we derived diploid pluripotent stem-cell lines from somatic cells of a newborn and, for the first time, an adult, a female with type 1 diabetes.

  14. Genetic and Epigenetic Changes in Somatic Hybrid Introgression Lines Between Wheat and Tall Wheatgrass

    PubMed Central

    Liu, Shuwei; Li, Fei; Kong, Lina; Sun, Yang; Qin, Lumin; Chen, Suiyun; Cui, Haifeng; Huang, Yinghua; Xia, Guangmin

    2015-01-01

    Broad phenotypic variations were induced in derivatives of an asymmetric somatic hybridization of bread wheat (Triticum aestivum) and tall wheatgrass (Thinopyrum ponticum Podp); however, how these variations occurred was unknown. We explored the nature of these variations by cytogenetic assays and DNA profiling techniques to characterize six genetically stable somatic introgression lines. Karyotyping results show the six lines similar to their wheat parent, but GISH analysis identified the presence of a number of short introgressed tall wheatgrass chromatin segments. DNA profiling revealed many genetic and epigenetic differences, including sequences deletions, altered regulation of gene expression, changed patterns of cytosine methylation, and the reactivation of retrotransposons. Phenotypic variations appear to result from altered repetitive sequences combined with the epigenetic regulation of gene expression and/or retrotransposon transposition. The extent of genetic and epigenetic variation due to the maintenance of parent wheat cells in tissue culture was assessed and shown to be considerably lower than had been induced in the introgression lines. Asymmetric somatic hybridization provides appropriate material to explore the nature of the genetic and epigenetic variations induced by genomic shock. PMID:25670745

  15. Metabolic Profiling of Somatic Tissues from Monochamus alternatus (Coleoptera: Cerambycidae) Reveals Effects of Irradiation on Metabolism

    PubMed Central

    Qu, Liangjian; Wang, Lijuan; Wang, Qinghua; Wang, Yuzhu; Zhang, Yongan

    2014-01-01

    A high-level of sexual sterility is of importance for the sterile insect technique (SIT). However, the use of high-dose-intensity gamma radiation to induce sterility has negative impacts not only on reproductive cells but also on somatic cells. In this study, we investigated the metabolite differences in somatic tissues between non-irradiated, 20-Gy-irradiated, and 40-Gy-irradiated male Monochamus alternatus, an important vector of the pathogenic nematode, Bursaphelenchus xylophilus, which kills Asian pines. The results showed that metabolite levels changed moderately in the 20-Gy samples but were markedly altered in the 40-Gy samples compared with the non-irradiated samples. Twenty-six and 53 metabolites were disturbed by 20-Gy and 40-Gy radiation, respectively. Thirty-six metabolites were found to be markedly altered in the 40-Gy samples but were not changed significantly in the 20-Gy samples. The comprehensive metabolomic disorders induced by 40-Gy radiation dysregulated six metabolic pathways involved in the life process. The findings presented in this manuscript will contribute to our knowledge of the characteristic metabolic changes associated with gamma-radiation-induced damage to somatic cells and will allow for better exploration of the SIT for the control of this target pest. PMID:24937685

  16. Genetic and epigenetic changes in somatic hybrid introgression lines between wheat and tall wheatgrass.

    PubMed

    Liu, Shuwei; Li, Fei; Kong, Lina; Sun, Yang; Qin, Lumin; Chen, Suiyun; Cui, Haifeng; Huang, Yinghua; Xia, Guangmin

    2015-04-01

    Broad phenotypic variations were induced in derivatives of an asymmetric somatic hybridization of bread wheat (Triticum aestivum) and tall wheatgrass (Thinopyrum ponticum Podp); however, how these variations occurred was unknown. We explored the nature of these variations by cytogenetic assays and DNA profiling techniques to characterize six genetically stable somatic introgression lines. Karyotyping results show the six lines similar to their wheat parent, but GISH analysis identified the presence of a number of short introgressed tall wheatgrass chromatin segments. DNA profiling revealed many genetic and epigenetic differences, including sequences deletions, altered regulation of gene expression, changed patterns of cytosine methylation, and the reactivation of retrotransposons. Phenotypic variations appear to result from altered repetitive sequences combined with the epigenetic regulation of gene expression and/or retrotransposon transposition. The extent of genetic and epigenetic variation due to the maintenance of parent wheat cells in tissue culture was assessed and shown to be considerably lower than had been induced in the introgression lines. Asymmetric somatic hybridization provides appropriate material to explore the nature of the genetic and epigenetic variations induced by genomic shock. PMID:25670745

  17. 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.

  18. 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

  19. Early-stage epigenetic modification during somatic cell reprogramming by Parp1 and Tet2.

    PubMed

    Doege, Claudia A; Inoue, Keiichi; Yamashita, Toru; Rhee, David B; Travis, Skylar; Fujita, Ryousuke; Guarnieri, Paolo; Bhagat, Govind; Vanti, William B; Shih, Alan; Levine, Ross L; Nik, Sara; Chen, Emily I; Abeliovich, Asa

    2012-08-30

    Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by using the pluripotency factors Oct4, Sox2, Klf4 and c-Myc (together referred to as OSKM). iPSC reprogramming erases somatic epigenetic signatures—as typified by DNA methylation or histone modification at silent pluripotency loci—and establishes alternative epigenetic marks of embryonic stem cells (ESCs). Here we describe an early and essential stage of somatic cell reprogramming, preceding the induction of transcription at endogenous pluripotency loci such as Nanog and Esrrb. By day 4 after transduction with OSKM, two epigenetic modification factors necessary for iPSC generation, namely poly(ADP-ribose) polymerase-1 (Parp1) and ten-eleven translocation-2 (Tet2), are recruited to the Nanog and Esrrb loci. These epigenetic modification factors seem to have complementary roles in the establishment of early epigenetic marks during somatic cell reprogramming: Parp1 functions in the regulation of 5-methylcytosine (5mC) modification, whereas Tet2 is essential for the early generation of 5-hydroxymethylcytosine (5hmC) by the oxidation of 5mC (refs 3,4). Although 5hmC has been proposed to serve primarily as an intermediate in 5mC demethylation to cytosine in certain contexts, our data, and also studies of Tet2-mutant human tumour cells, argue in favour of a role for 5hmC as an epigenetic mark distinct from 5mC. Consistent with this, Parp1 and Tet2 are each needed for the early establishment of histone modifications that typify an activated chromatin state at pluripotency loci, whereas Parp1 induction further promotes accessibility to the Oct4 reprogramming factor. These findings suggest that Parp1 and Tet2 contribute to an epigenetic program that directs subsequent transcriptional induction at pluripotency loci during somatic cell reprogramming. PMID:22902501

  20. [Somatic chromosome mutagenesis in residents of Ukraine exposed to ionizing radiation in different periods after the Chernobyl accident].

    PubMed

    Pilinskaia, M A; Dybskiĭ, S S; Shemetun, E V; Dybskaia, E B

    2011-01-01

    The authors summarize results of 25-year selective cytogenetic monitoring of the priority groups in different periods after the Chernobyl accident. The increase in intensity of somatic chromosome mutagenesis in exposed individuals as a result of both targeted and non-targeted radiation-induced cytogenetic effects has been confirmed including delayed, transmissible, hidden chromosome instability and the bystander effect.