Hemoglobin promotes somatic embryogenesis in peanut cultures.

PubMed

Jayabalan, N; Anthony, P; Davey, M R; Power, J B; Lowe, K C

2004-02-01

Critical parameters influencing somatic embryogenesis include growth regulators and oxygen supply. Consequently, the present investigation has focused on optimization of a somatic embryogenic system for peanut (Arachis hypogaea L.) through media supplementation with the auxin, picloram. The latter at 30 mg L(-1) was optimal for inducing regeneration of somatic embryos from cultured explants of zygotic embryos. In contrast, somatic embryogenesis did not occur in the absence of this growth regulator. An assessment has also been made of the beneficial effect on somatic embryogenesis and plant regeneration of the commercial hemoglobin (Hb) solution, Erythrogen. Hemoglobin at 1:50 and 1:100 (v:v) stimulated increases in mean fresh weight (up to a maximum of 57% over control), mean number of explants producing somatic embryos (15%) and mean number of somatic embryos per explant (29%).

Somatic embryogenesis from leaf explants of Australian fan flower, Scaevola aemula R. Br.

PubMed

Wang, Y-H; Bhalla, P L

2004-01-01

Somatic embryogenesis from leaf explants of Scaevola aemula R. Br. was achieved. Somatic embryos were induced from explants cultured on MS medium supplemented with 0.2 mg/ 2,4-dichlorophenoxyacetic acid and 0.2-0.5 mg/l 6-benzylaminopurine (BAP). Various developmental stages of somatic embryos were found on this medium-from globular embryos to germinated embryos. The transfer of globular embryos to MS medium containing 0.5 mg/l BAP resulted in a high frequency of shoot regeneration. Leaf explants cultured on MS medium containing different combinations of BAP and alpha-naphthaleneacetic acid formed adventitious shoots and roots. Histological examination confirmed the process of somatic embryogenesis. Induction of somatic embryogenesis in Scaevola provides a system for studying embryogenesis in Australian native plants and will facilitate the improvement of these plants using genetic transformation techniques.

Shoot regeneration and somatic embryogenesis from needles of redwood (Sequoia sempervirens (D.Don.) Endl.).

PubMed

Liu, Cuiqiong; Xia, Xinli; Yin, Weilun; Huang, Lichun; Zhou, Jianghong

2006-07-01

A rapid and effective system of somatic embryogenesis and organogenesis from the in vitro needles of redwood (Sequoia sempervirens (D.Don.) Endl.) had been established. The influences of plant growth regulators (PGRs) and days of seedlings in vitro on adventitious bud regeneration and somatic embryogenesis were studied. The process of somatic embryo formation was also observed. The results showed that embryogenic callus was induced and proliferated on Schenk and Hildebrandt (SH) medium with BA (0.5 mg/l), KT (0.5 mg/l) and IBA (1.0 mg/l). SH medium containing BA (0.5 mg/l), KT (0.2 mg/l) and IBA (0.2 mg/l) effectively promoted adventitious bud regeneration. The highest frequency (66.3%) of direct somatic embryogenesis was obtained in the combination of BA (0.5 mg/l) and IBA (0.5 mg/l). The optimal days of seedling in vitro for adventitious bud and somatic embryogenesis were 30 days and 30-40 days, respectively. The developments of somatic embryos were similar to that of zygotic embryogenesis. The result of histocytological studies indicated that proteins were gradually accumulated in the process of somatic embryo formation and there were two peaks of starch grains accumulation that one was in the embryogenic callus and the other was in the globular embryos. These results indicated that starch and protein were closely related with the energy supply and the molecular base of somatic embryogenesis, respectively.

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

Bottlenecks in bog pine multiplication by somatic embryogenesis and their visualization with the environmental scanning electron microscope.

PubMed

Vlašínová, Helena; Neděla, Vilem; Đorđević, Biljana; Havel, Ladislav

2017-07-01

Somatic embryogenesis (SE) is an important biotechnological technique used for the propagation of many pine species in vitro. However, in bog pine, one of the most endangered tree species in the Czech Republic, limitations were observed, which negatively influenced the development and further germination of somatic embryos. Although initiation frequency was very low-0.95 %, all obtained cell lines were subjected to maturation. The best responding cell line (BC1) was used and subjected to six different variants of the maturation media. The media on which the highest number of early-precotyledonary/cotyledonary somatic embryos was formed was supplemented with 121 μM abscisic acid (ABA) and with 6 % maltose. In the end of maturation experiments, different abnormalities in formation of somatic embryos were observed. For visualization and identification of abnormalities in meristem development during proliferation and maturation processes, the environmental scanning electron microscope was used. In comparison to the classical light microscope, the non-commercial environmental scanning electron microscope AQUASEM II has been found as a very useful tool for the quick recognition of apical meristem disruption and abnormal development. To our knowledge, this is the first report discussing somatic embryogenesis in bog pine. Based on this observation, the cultivation procedure could be enhanced and the method for SE of bog pine optimized.

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

PubMed Central

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

2011-01-01

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

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.

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.

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.

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

PubMed Central

2012-01-01

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

The BABY BOOM Transcription Factor Activates the LEC1-ABI3-FUS3-LEC2 Network to Induce Somatic Embryogenesis1[OPEN

PubMed Central

Weemen, Mieke

2017-01-01

Somatic embryogenesis is an example of induced cellular totipotency, where embryos develop from vegetative cells rather than from gamete fusion. Somatic embryogenesis can be induced in vitro by exposing explants to growth regulators and/or stress treatments. The BABY BOOM (BBM) and LEAFY COTYLEDON1 (LEC1) and LEC2 transcription factors are key regulators of plant cell totipotency, as ectopic overexpression of either transcription factor induces somatic embryo formation from Arabidopsis (Arabidopsis thaliana) seedlings without exogenous growth regulators or stress treatments. Although LEC and BBM proteins regulate the same developmental process, it is not known whether they function in the same molecular pathway. We show that BBM transcriptionally regulates LEC1 and LEC2, as well as the two other LAFL genes, FUSCA3 (FUS3) and ABSCISIC ACID INSENSITIVE3 (ABI3). LEC2 and ABI3 quantitatively regulate BBM-mediated somatic embryogenesis, while FUS3 and LEC1 are essential for this process. BBM-mediated somatic embryogenesis is dose and context dependent, and the context-dependent phenotypes are associated with differential LAFL expression. We also uncover functional redundancy for somatic embryogenesis among other Arabidopsis BBM-like proteins and show that one of these proteins, PLETHORA2, also regulates LAFL gene expression. Our data place BBM upstream of other major regulators of plant embryo identity and totipotency. PMID:28830937

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.

Desiccation Treatment and Endogenous IAA Levels Are Key Factors Influencing High Frequency Somatic Embryogenesis in Cunninghamia lanceolata (Lamb.) Hook

PubMed Central

Zhou, Xiaohong; Zheng, Renhua; Liu, Guangxin; Xu, Yang; Zhou, Yanwei; Laux, Thomas; Zhen, Yan; Harding, Scott A.; Shi, Jisen; Chen, Jinhui

2017-01-01

Cunninghamia lanceolata (Lamb.) Hook (Chinese fir) is an important tree, commercially and ecologically, in southern China. The traditional regenerating methods are based on organogenesis and cutting propagation. Here, we report the development of a high-frequency somatic embryogenesis (SE) regeneration system synchronized via a liquid culture from immature zygotic embryos. Following synchronization, PEM II cell aggregates were developmentally equivalent in appearance to cleaved zygotic embryos. Embryo and suspensor growth and subsequent occurrence of the apical and then the cotyledonary meristems were similar for zygotic and SE embryo development. However, SE proembryos exhibited a more reddish coloration than zygotic proembryos, and SE embryos were smaller than zygotic embryos. Mature somatic embryos gave rise to plantlets on hormone-free medium. For juvenile explants, low concentrations of endogenous indole-3-acetic acid in initial explants correlated with improved proembryogenic mass formation, and high SE competency. Analysis of karyotypes and microsatellites detected no major genetic variation in the plants regenerated via SE, and suggest a potential in the further development of this system as a reliable methodology for true-to-type seedling production. Treatment with polyethylene glycol (PEG) and abscisic acid (ABA) were of great importance to proembryo formation and complemented each other. ABA assisted the growth of embryonal masses, whereas PEG facilitated the organization of the proembryo-like structures. SOMATIC EMBRYOGENESIS RECEPTOR KINASE SERK) and the WUSCHEL homeobox (WOX) transcription factor served as molecular markers during early embryogenesis. Our results show that ClSERKs are conserved and redundantly expressed during SE. SERK and WOX transcript levels were highest during development of the proembryos and lowest in developed embryos. ClWOX13 expression correlates with the critical transition from proembryogenic masses to proembryos. Both SERK and WOX expression reveal their applicability in Chinese fir as markers of early embryogenesis. Overall, the findings provided evidence for the potential of this system in high fidelity Chinese fir seedlings production. Also, SE modification strategies were demonstrated and could be applied in other conifer species on the basis of our hormonal, morphological and molecular analyses. PMID:29259612

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.

Chemical Compositions, Somatic Embryogenesis, and Somaclonal Variation in Cumin

PubMed Central

Tohidfar, Masoud; Sadat Noori, Seyed Ahmad; Izadi Darbandi, Ali; Rao, Rosa

2017-01-01

This is the first report evaluating the relationship between the chemical compositions of cumin seeds (based on the analysis of the content of catalase, ascorbate peroxidase, proline, protein, terpenic compounds, alcohol/phenols, aldehydes, and epoxides) and the induction efficiency of somatic embryogenesis in two Iranian superior cumin landraces (Golestan and North Khorasan). Cotyledons isolated from Golestan landrace seeds cultivated on MS medium supplemented with 0.1 mg/L kinetin proved to be the best primary explant for the induction of somatic embryogenesis as well as the regeneration of the whole plantlet. Results indicated that different developmental stages of somatic embryos were simultaneously observed on a callus with embryogenic potential. The high content of catalase, ascorbate peroxidase, proline, and terpenic hydrocarbons and low content of alcoholic and phenolic compositions had a stimulatory effect on somatic embryogenesis. Band patterns of RAPD markers in regenerated plants were different from those of the mother plants. This may be related to somaclonal variations or pollination system of cumin. Generally, measurement of chemical compositions can be used as a marker for evaluating the occurrence of somatic embryogenesis in cumin. Also, somaclonal variations of regenerated plants can be applied by the plant breeders in breeding programs. PMID:29234682

Inducible somatic embryogenesis in Theobroma cacao achieved using the DEX-activatable transcription factor-glucocorticoid receptor fusion.

PubMed

Shires, Morgan E; Florez, Sergio L; Lai, Tina S; Curtis, Wayne R

2017-11-01

To carry out mass propagation of superior plants to improve agricultural and silvicultural production though advancements in plant cell totipotency, or the ability of differentiated somatic plant cells to regenerate an entire plant. The first demonstration of a titratable control over somatic embryo formation in a commercially relevant plant, Theobroma cacao (Chocolate tree), was achieved using a dexamethasone activatable chimeric transcription factor. This four-fold enhancement in embryo production rate utilized a glucocorticoid receptor fused to an embryogenic transcription factor LEAFY COTYLEDON 2. Where previous T. cacao somatic embryogenesis has been restricted to dissected flower parts, this construct confers an unprecedented embryogenic potential to leaves. Activatable chimeric transcription factors provide a means for elucidating the regulatory cascade associated with plant somatic embryogenesis towards improving its use for somatic regeneration of transgenics and plant propagation.

Somatic Embryogenesis in Lisianthus (Eustoma russellianum Griseb.).

PubMed

Ruffoni, Barbara; Bassolino, Laura

2016-01-01

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

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'.

Synchronization of Somatic Embryogenesis in Date Palm Suspension Culture Using Abscisic Acid.

PubMed

Alwael, Hussain A; Naik, Poornananda M; Al-Khayri, Jameel M

2017-01-01

Somatic embryogenesis is considered the most effective method for commercial propagation of date palm. However, the limitation of obtaining synchronized development of somatic embryos remains an impediment. The synchronization of somatic embryo development is ideal for the applications to produce artificial seeds. Abscisic acid (ABA) is associated with stress response and influences in vitro growth and development. This chapter describes an effective method to achieve synchronized development of somatic embryos in date palm cell suspension culture. Among the ABA concentrations tested (0, 1, 10, 50, 100 μM), the best synchronized growth was obtained in response to 50-100 μM. Here we provide a comprehensive protocol for in vitro plant regeneration of date palm starting with shoot-tip explant, callus initiation and growth, cell suspension establishment, embryogenesis synchronization with ABA treatment, somatic embryo germination, and rooting as well as acclimatized plantlet establishment.

Flow cytometric and morphological analyses of Pinus pinaster somatic embryogenesis.

PubMed

Marum, Liliana; Loureiro, João; Rodriguez, Eleazar; Santos, Conceição; Oliveira, M Margarida; Miguel, Célia

2009-09-25

An approach combining morphological profiling and flow cytometric analysis was used to assess genetic stability during the several steps of somatic embryogenesis in Pinus pinaster. Embryogenic cell lines of P. pinaster were established from immature zygotic embryos excised from seeds obtained from open-pollinated trees. During the maturation stage, phenotype of somatic embryos was characterized as being either normal or abnormal. Based upon the prevalent morphological traits, different types of abnormal embryos underwent further classification and quantification. Nuclear DNA content of maritime pine using the zygotic embryos was estimated to be 57.04 pg/2C, using propidium iodide flow cytometry. According to the same methodology, no significant differences (P< or =0.01) in DNA ploidy were detected among the most frequently observed abnormal phenotypes, embryogenic cell lines, zygotic and normal somatic embryos, and somatic embryogenesis-derived plantlets. Although the differences in DNA ploidy level do not exclude the occurrence of a low level of aneuploidy, the results obtained point to the absence of major changes in ploidy level during the somatic embryogenesis process of this economically important species. Therefore, our primary goal of true-to-typeness was assured at this level.

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.

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

PubMed

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

2003-10-01

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

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

Unfertilized ovary: a novel explant for coconut (Cocos nucifera L.) somatic embryogenesis.

PubMed

Perera, Prasanthi I P; Hocher, Valerie; Verdeil, Jean Luc; Doulbeau, Sylvie; Yakandawala, Deepthi M D; Weerakoon, L Kaushalya

2007-01-01

Unfertilized ovaries isolated from immature female flowers of coconut (Cocos nucifera L.) were tested as a source of explants for callogenesis and somatic embryogenesis. The correct developmental stage of ovary explants and suitable in vitro culture conditions for consistent callus production were identified. The concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) and activated charcoal was found to be critical for callogenesis. When cultured in a medium containing 100 microM 2,4-D and 0.1% activated charcoal, ovary explants gave rise to 41% callusing. Embryogenic calli were sub-cultured into somatic embryogenesis induction medium containing 5 microM abscisic acid, followed by plant regeneration medium (with 5 microM 6-benzylaminopurine). Many of the somatic embryos formed were complete with shoot and root poles and upon germination they gave rise to normal shoots. However, some abnormal developments were also observed. Flow cytometric analysis revealed that all the calli tested were diploid. Through histological studies, it was possible to study the sequence of the events that take place during somatic embryogenesis including orientation, polarization and elongation of the embryos.

Problems and potentialities of cultured plant cells in retrospect and prospect

NASA Technical Reports Server (NTRS)

Steward, F. C.; Krikorian, A. D.

1979-01-01

The past, present and expected future accomplishments and limitations of plant cell and tissue culture are reviewed. Consideration is given to the pioneering insights of Haberlandt in 1902, the development of culture techniques, and past work on cell division, cell and tissue growth and development, somatic embryogenesis, and metabolism and respiration. Current activity in culture media and technique development for plant regions, organs, tissues, cells, protoplasts, organelles and embryos, totipotency, somatic embryogenesis and clonal propagation under normal and space conditions, biochemical potentialities, and genetic engineering is surveyed. Prospects for the investigation of the induced control of somatic cell division, the division of isolated protoplasts, the improvement of haploid cell cultures, liquid cultures for somatic embryogenesis, and the genetic control of development are outlined.

Somatic embryos from culture ovules of polyembryonic Mangifera indica L.

PubMed

Litz, R E; Knight, R L; Gazit, S

1982-12-01

Ovules were aseptically removed from 2 month old fruits of 9 naturally polyembryonic cultivars and 1 monoembryonic cultivar of mango (Mangifera indica L.). Ovules were placed into culture on solid Murashige and Skoog medium that had been modified by the addition of half strength major salts and chelated iron, 6% sucrose, 400 mg/l glutamine, 100 mg/l ascorbic acid with or without the following growth regulators: 20% (v/v) CW, 1 or 2 mg/1 BA. Somatic embryogenesis occurred from the nucellus excised from the ovules of 5 of the naturally polyembryonic cultivars after 1-2 months in culture. Somatic embryogenesis was not apparently affected by the growth regulator composition of the media; however, efficient somatic embryogenesis only occurred in liquid containing 20% CW.

De novo assembly and characterization of global transcriptome of coconut palm (Cocos nucifera L.) embryogenic calli using Illumina paired-end sequencing.

PubMed

Rajesh, M K; Fayas, T P; Naganeeswaran, S; Rachana, K E; Bhavyashree, U; Sajini, K K; Karun, Anitha

2016-05-01

Production and supply of quality planting material is significant to coconut cultivation but is one of the major constraints in coconut productivity. Rapid multiplication of coconut through in vitro techniques, therefore, is of paramount importance. Although somatic embryogenesis in coconut is a promising technique that will allow for the mass production of high quality palms, coconut is highly recalcitrant to in vitro culture. In order to overcome the bottlenecks in coconut somatic embryogenesis and to develop a repeatable protocol, it is imperative to understand, identify, and characterize molecular events involved in coconut somatic embryogenesis pathway. Transcriptome analysis (RNA-Seq) of coconut embryogenic calli, derived from plumular explants of West Coast Tall cultivar, was undertaken on an Illumina HiSeq 2000 platform. After de novo transcriptome assembly and functional annotation, we have obtained 40,367 transcripts which showed significant BLASTx matches with similarity greater than 40 % and E value of ≤10(-5). Fourteen genes known to be involved in somatic embryogenesis were identified. Quantitative real-time PCR (qRT-PCR) analyses of these 14 genes were carried in six developmental stages. The result showed that CLV was upregulated in the initial stage of callogenesis. Transcripts GLP, GST, PKL, WUS, and WRKY were expressed more in somatic embryo stage. The expression of SERK, MAPK, AP2, SAUR, ECP, AGP, LEA, and ANT were higher in the embryogenic callus stage compared to initial culture and somatic embryo stages. This study provides the first insights into the gene expression patterns during somatic embryogenesis in coconut.

Plant regeneration via direct somatic embryogenesis from leaf explants of Tolumnia Louise Elmore 'Elsa'.

PubMed

Shen, Hui-Ju; Chen, Jen-Tsung; Chung, Hsiao-Hang; Chang, Wei-Chin

2018-01-22

Tolumnia genus (equitant Oncidium) is a group of small orchids with vivid flower color. Thousands of hybrids have been registered on Royal Horticulture Society and showed great potential for ornamental plant market. The aim of this study is to establish an efficient method for in vitro propagation. Leaf explants taken from in vitro-grown plants were used to induce direct somatic embryogenesis on a modified 1/2 MS medium supplemented with five kinds of cytokinins, 2iP, BA, kinetin, TDZ and zeatin at 0.3, 1 and 3 mg l -1 in darkness. TDZ at 3 mg l -1 gave the highest percentage of explants with somatic globular embryos after 90 days of culture. It was found that 2,4-D and light regime highly retarded direct somatic embryogenesis and showed 95-100% of explant browning. Histological observations revealed that the leaf cells divided into meristematic cells firstly, followed by somatic proembryos, and then somatic globular embryos. Eventually, somatic embryos developed a bipolar structure with the shoot apical meristem and the root meristem. Scanning electron microscopy observations showed that the direct somatic embryogenesis from leaf explants was asynchronously. The somatic embryos were found on the leaf tip, the adaxial surface and also the mesophyll through a cleft, and it reflected the heterogeneity of the explant. The 90-day-old globular embryos were detached from the parent explants and transferred onto a hormone-free 1/2 MS medium in light condition for about 1 month to obtain 1-cm-height plantlets. After another 3 months for growth, the plantlets were potted with Sphagnum moss and were acclimatized in a shaded greenhouse. After 1 month of culture, the survival rate was 100%. In this report, a protocol for efficient regenerating a Tolumnia orchid, Louise Elmore 'Elsa', was established via direct somatic embryogenesis and might reveal an alternative approach for mass propagation of Tolumnia genus in orchid industry.

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

Yield performance of cacao propagated by somatic embryogenesis and grafting

USDA-ARS?s Scientific Manuscript database

Twelve cacao (Theobroma cacao) clones propagated by grafting and somatic embryogenesis and grown on an Ultisol soil were evaluated for five years under intensive management at Corozal, Puerto Rico. Preliminary data showed no significant differences between propagation methods for yield of dry beans ...

In vitro somatic embryogenesis and plant regeneration of cassava.

PubMed

Szabados, L; Hoyos, R; Roca, W

1987-06-01

An efficient and reproducible plant regeneration system, initiated in somatic tissues, has been devised for cassava (Manihot esculenta Crantz). Somatic embryogenesis has been induced from shoot tips and immature leaves of in vitro shoot cultures of 15 cassava genotypes. Somatic embryos developed directly on the explants when cultured on a medium containing 4-16 mg/l 2,4-D. Differences were observed with respect to the embryogenic capacity of the explants of different varieties. Secondary embryogenesis has been induced by subculture on solid or liquid induction medium. Long term cultures were established and maintained for up to 18 months by repeated subculture of the proliferating somatic embryos. Plantlets developed from primary and secondary embryos in the presence of 0.1 mg/l BAP, 1mg/l GA3, and 0.01 mg/l 2,4-D. Regenerated plants were transferred to the field, and were grown to maturity.

The Theobroma cacao B3 domain transcription factor TcLEC2 plays a duel role in control of embryo development and maturation.

PubMed

Zhang, Yufan; Clemens, Adam; Maximova, Siela N; Guiltinan, Mark J

2014-04-24

The Arabidopsis thaliana LEC2 gene encodes a B3 domain transcription factor, which plays critical roles during both zygotic and somatic embryogenesis. LEC2 exerts significant impacts on determining embryogenic potential and various metabolic processes through a complicated genetic regulatory network. An ortholog of the Arabidopsis Leafy Cotyledon 2 gene (AtLEC2) was characterized in Theobroma cacao (TcLEC2). TcLEC2 encodes a B3 domain transcription factor preferentially expressed during early and late zygotic embryo development. The expression of TcLEC2 was higher in dedifferentiated cells competent for somatic embryogenesis (embryogenic calli), compared to non-embryogenic calli. Transient overexpression of TcLEC2 in immature zygotic embryos resulted in changes in gene expression profiles and fatty acid composition. Ectopic expression of TcLEC2 in cacao leaves changed the expression levels of several seed related genes. The overexpression of TcLEC2 in cacao explants greatly increased the frequency of regeneration of stably transformed somatic embryos. TcLEC2 overexpressing cotyledon explants exhibited a very high level of embryogenic competency and when cultured on hormone free medium, exhibited an iterative embryogenic chain-reaction. Our study revealed essential roles of TcLEC2 during both zygotic and somatic embryo development. Collectively, our evidence supports the conclusion that TcLEC2 is a functional ortholog of AtLEC2 and that it is involved in similar genetic regulatory networks during cacao somatic embryogenesis. To our knowledge, this is the first detailed report of the functional analysis of a LEC2 ortholog in a species other then Arabidopsis. TcLEC2 could potentially be used as a biomarker for the improvement of the SE process and screen for elite varieties in cacao germplasm.

[Direct and indirect somatic embryogenesis in Freesia refracta].

PubMed

Wang, L; Duan, X G; Hao, S

1999-06-01

Somatic embryogenesis can be induced in tissue cultures of Freesia refracta either directly from the epidermal cells of explant, or indirectly via intervening callus. In direct pathway, somatic embryos were in contact with maternal tissue in a suspensor-like structure. In indirect pathway, the explants first proliferacted to give rise to calluses before embryoids were induced. The two sorts of calluses were defined to embryogenic callus and non-embryogenic callus according to producing of somatic embryos. An indirect somatic embryo is developed from a pre-embryogenically determined cell. This kind of somatic embryo has no suspensor structure instead of a complex with maternal tissue. Somatic embryos have their own vascular tissues, and can develop new plantlets independently.

Somatic embryogenesis from corolla tubes of interspecific amphiploids between cultivated sunflower (Helianthus annuus L.) and its wild species

USDA-ARS?s Scientific Manuscript database

Somatic embryogenesis in vitro provides an efficient means of plant multiplication, facilitating sunflower improvement and germplasm innovation. In the present study, using interspecific amphiploids (2n=4x=68) between cultivated sunflower and wild perennial Helianthus species as explant donors, soma...

Preliminary molecular detection of the somatic embryogenesis receptor-like kinase (VpSERK) and knotted-like homeobox (VpKNOX1) genes during in vitro morphogenesis of Vanilla planifolia Jacks.

PubMed

Ramírez-Mosqueda, Marco A; Iglesias-Andreu, Lourdes G; Sáenz, Luis; Córdova, Iván

2018-02-01

This work aimed to evaluate the embryogenic competence of different tissues from different stages (friable callus, bud-regenerating callus, and whole buds) of Vanilla planifolia , through the molecular detection of the somatic embryogenesis receptor-like kinase ( VpSERK ) and knotted-like homeobox ( VpKNOX1 ) genes. RNA was extracted with Trizol ® , cDNA was obtained, and the studied transcripts were amplified. Using non-specific primers, VpSERK and VpSTM gene expression was detected in the three stages evaluated. This study might contribute to providing an explanation for the recalcitrance of this Vanilla species to somatic embryogenesis.

Stage-specific regulation of four HD-ZIP III transcription factors during polar pattern formation in Larix leptolepis somatic embryos.

PubMed

Li, Shui-gen; Li, Wan-feng; Han, Su-ying; Yang, Wen-hua; Qi, Li-wang

2013-06-15

Polar auxin transport provides a developmental signal for cell fate specification during somatic embryogenesis. Some members of the HD-ZIP III transcription factors participate in regulation of auxin transport, but little is known about this regulation in somatic embryogenesis. Here, four HD-ZIP III homologues from Larix leptolepis were identified and designated LaHDZ31, 32, 33 and 34. The occurrence of a miR165/166 target sequence in all four cDNA sequences indicated that they might be targets of miR165/166. Identification of the cleavage products of LaHDZ31 and LaHDZ32 in vivo confirmed that they were regulated by miRNA. Their mRNA accumulation patterns during somatic embryogenesis and the effects of 1-N-naphthylphthalamic acid (NPA) on their transcript levels and somatic embryo maturation were investigated. The results showed that the four genes had higher transcript levels at mature stages than at the proliferation stage, and that NPA treatment down-regulated the mRNA abundance of LaHDZ31, 32 and 33 at cotyledonary embryo stages, but had no effect on the mRNA abundance of LaHDZ34. We concluded that these four members of Larix HD-ZIP III family might participate in polar auxin transport and the development of somatic embryos, providing new insights into the regulatory mechanisms of somatic embryogenesis. Copyright © 2013 Elsevier B.V. All rights reserved.

Normalizing gene expression by quantitative PCR during somatic embryogenesis in two representative conifer species: Pinus pinaster and Picea abies.

PubMed

de Vega-Bartol, José J; Santos, Raquen Raissa; Simões, Marta; Miguel, Célia M

2013-05-01

Suitable internal control genes to normalize qPCR data from different stages of embryo development and germination were identified in two representative conifer species. Clonal propagation by somatic embryogenesis has a great application potentiality in conifers. Quantitative PCR (qPCR) is widely used for gene expression analysis during somatic embryogenesis and embryo germination. No single reference gene is universal, so a systematic characterization of endogenous genes for concrete conditions is fundamental for accuracy. We identified suitable internal control genes to normalize qPCR data obtained at different steps of somatic embryogenesis (embryonal mass proliferation, embryo maturation and germination) in two representative conifer species, Pinus pinaster and Picea abies. Candidate genes included endogenous genes commonly used in conifers, genes previously tested in model plants, and genes with a lower variation of the expression along embryo development according to genome-wide transcript profiling studies. Three different algorithms were used to evaluate expression stability. The geometric average of the expression values of elongation factor-1α, α-tubulin and histone 3 in P. pinaster, and elongation factor-1α, α-tubulin, adenosine kinase and CAC in P. abies were adequate for expression studies throughout somatic embryogenesis. However, improved accuracy was achieved when using other gene combinations in experiments with samples at a single developmental stage. The importance of studies selecting reference genes to use in different tissues or developmental stages within one or close species, and the instability of commonly used reference genes, is highlighted.

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

PubMed

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

2014-09-01

Maritime pine somatic embryos (SEs) require a reduction in water availability (high gellan gum concentration in the maturation medium) to reach the cotyledonary stage. This key switch, reported specifically for pine species, is not yet well understood. To facilitate the use of somatic embryogenesis for mass propagation of conifers, we need a better understanding of embryo development. Comparison of both transcriptome (Illumina RNA sequencing) and proteome [two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with mass spectrometry (MS) identification] of immature SEs, cultured on either high (9G) or low (4G) gellan gum concentration, was performed, together with analysis of water content, fresh and dry mass, endogenous abscisic acid (ABA; gas chromatography-MS), soluble sugars (high-pressure liquid chromatography), starch and confocal laser microscope observations. This multiscale, integrated analysis was used to unravel early molecular and physiological events involved in SE development. Under unfavorable conditions (4G), the glycolytic pathway was enhanced, possibly in relation to cell proliferation that may be antagonistic to SE development. Under favorable conditions (9G), SEs adapted to culture constraint by activating specific protective pathways, and ABA-mediated molecular and physiological responses promoting embryo development. Our results suggest that on 9G, germin-like protein and ubiquitin-protein ligase could be used as predictive markers of SE development, whereas protein phosphatase 2C could be a biomarker for culture adaptive responses. This is the first characterization of early molecular mechanisms involved in the development of pine SEs following an increase in gellan gum concentration in the maturation medium, and it is also the first report on somatic embryogenesis in conifers combining transcriptomic and proteomic datasets. © 2014 Scandinavian Plant Physiology Society.

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.

Micropropagation of Iris sp.

PubMed

Jevremović, Slađana; Jeknić, Zoran; Subotić, Angelina

2013-01-01

Irises are perennial plants widely used as ornamental garden plants or cut flowers. Some species accumulate secondary metabolites, making them highly valuable to the pharmaceutical and perfume industries. Micropropagation of irises has successfully been accomplished by culturing zygotic embryos, different flower parts, and leaf base tissues as starting explants. Plantlets are regenerated via somatic embryogenesis, organogenesis, or both processes at the same time depending on media composition and plant species. A large number of uniform plants are produced by somatic embryogenesis, however, some species have decreased morphogenetic potential overtime. Shoot cultures obtained by organogenesis can be multiplied for many years. Somatic embryogenic tissue can be reestablished from leaf bases of in vitro-grown shoots. The highest number of plants can be obtained by cell suspension cultures. This chapter describes effective in vitro plant regeneration protocols for Iris species from different types of explants by somatic embryogenesis and/or organogenesis suitable for the mass propagation of ornamental and pharmaceutical irises.

Somatic embryogenesis and plant regeneration of northern red oak (Quercus rubra L.)

Treesearch

G. Vengadesan; Paula M. Pijut

2009-01-01

A somatic embryogenesis protocol for plant regeneration of northern red oak (Quercus rubra) was established from immature cotyledon explants. Embryogenic callus cultures were induced on Murashige and Skoog medium (MS) containing 3% sucrose, 0.24% Phytagel™, and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) after 4 weeks of...

Embryonic origin of adult stem cells required for tissue homeostasis and regeneration

PubMed Central

Davies, Erin L; Lei, Kai; Seidel, Christopher W; Kroesen, Amanda E; McKinney, Sean A; Guo, Longhua; Robb, Sofia MC; Ross, Eric J; Gotting, Kirsten; Alvarado, Alejandro Sánchez

2017-01-01

Planarian neoblasts are pluripotent, adult somatic stem cells and lineage-primed progenitors that are required for the production and maintenance of all differentiated cell types, including the germline. Neoblasts, originally defined as undifferentiated cells residing in the adult parenchyma, are frequently compared to embryonic stem cells yet their developmental origin remains obscure. We investigated the provenance of neoblasts during Schmidtea mediterranea embryogenesis, and report that neoblasts arise from an anarchic, cycling piwi-1+ population wholly responsible for production of all temporary and definitive organs during embryogenesis. Early embryonic piwi-1+ cells are molecularly and functionally distinct from neoblasts: they express unique cohorts of early embryo enriched transcripts and behave differently than neoblasts in cell transplantation assays. Neoblast lineages arise as organogenesis begins and are required for construction of all major organ systems during embryogenesis. These subpopulations are continuously generated during adulthood, where they act as agents of tissue homeostasis and regeneration. DOI: http://dx.doi.org/10.7554/eLife.21052.001 PMID:28072387

Somatic embryogenesis in cell cultures of Glycine species.

PubMed

Gamborg, O L; Davis, B P; Stahlhut, R W

1983-08-01

This report describes the development of procedures for the production of somatic embryos in cell cultures of Glycine species including soybean. The conditions for callus induction and initiation of rapidly growing cell suspension cultures were defined. Methods for inducing embryogenesis were tested on 16 lines of several Glycine species and cultivars of soybean. The SB-26 Culture of a G. soja gave the best results and was used in the experiments. Embryogenesis required the presence of picloram or 2,4-D. AMO 1618, CCC, PP-333 and Ancymidol enhanced the embryogenesis frequency. Plants of the G. soja (SB-26) were grown to maturity from seed-derived shoot tips. Characteristics of the plants are discussed.

The role of arginine metabolic pathway during embryogenesis and germination in maritime pine (Pinus pinaster Ait.).

PubMed

Llebrés, María-Teresa; Pascual, María-Belén; Debille, Sandrine; Trontin, Jean-François; Harvengt, Luc; Avila, Concepción; Cánovas, Francisco M

2018-03-01

Vegetative propagation through somatic embryogenesis is critical in conifer biotechnology towards multivarietal forestry that uses elite varieties to cope with environmental and socio-economic issues. An important and still sub-optimal process during in vitro maturation of somatic embryos (SE) is the biosynthesis and deposition of storage proteins, which are rich in amino acids with high nitrogen (N) content, such as arginine. Mobilization of these N-rich proteins is essential for the germination and production of vigorous somatic seedlings. Somatic embryos accumulate lower levels of N reserves than zygotic embryos (ZE) at a similar stage of development. To understand the molecular basis for this difference, the arginine metabolic pathway has been characterized in maritime pine (Pinus pinaster Ait.). The genes involved in arginine metabolism have been identified and GFP-fusion constructs were used to locate the enzymes in different cellular compartments and clarify their metabolic roles during embryogenesis and germination. Analysis of gene expression during somatic embryo maturation revealed high levels of transcripts for genes involved in the biosynthesis and metabolic utilization of arginine. By contrast, enhanced expression levels were only observed during the last stages of maturation and germination of ZE, consistent with the adequate accumulation and mobilization of protein reserves. These results suggest that arginine metabolism is unbalanced in SE (simultaneous biosynthesis and degradation of arginine) and could explain the lower accumulation of storage proteins observed during the late stages of somatic embryogenesis.

Polyamine and ethylene biosynthesis in relation to somatic embryogenesis in carrot (Daucus carota L.) cell cultures

Treesearch

Subhash C. Minocha; Cheryl A. Robie; Akhtar J. Khan; Nancy S. Papa; Andrew I. Samuelsen; Rakesh Minocha

1990-01-01

Carrot cell cultures provide a model experimental system for the analysis of biochemical and molecular events associated with morphogenesis in plants (3, 4, 5, 14). Among the biochemical changes accompanying somatic embryogenesis in this tissue is an increased biosynthesis ofpolyamines (1, 2, 7, 10, 11, 13). A variety of inhibitors of polyamine biosynthetic enzymes...

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

USDA-ARS?s Scientific Manuscript database

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 ...

The Theobroma cacao B3 domain transcription factor TcLEC2 plays a duel role in control of embryo development and maturation

PubMed Central

2014-01-01

Background The Arabidopsis thaliana LEC2 gene encodes a B3 domain transcription factor, which plays critical roles during both zygotic and somatic embryogenesis. LEC2 exerts significant impacts on determining embryogenic potential and various metabolic processes through a complicated genetic regulatory network. Results An ortholog of the Arabidopsis Leafy Cotyledon 2 gene (AtLEC2) was characterized in Theobroma cacao (TcLEC2). TcLEC2 encodes a B3 domain transcription factor preferentially expressed during early and late zygotic embryo development. The expression of TcLEC2 was higher in dedifferentiated cells competent for somatic embryogenesis (embryogenic calli), compared to non-embryogenic calli. Transient overexpression of TcLEC2 in immature zygotic embryos resulted in changes in gene expression profiles and fatty acid composition. Ectopic expression of TcLEC2 in cacao leaves changed the expression levels of several seed related genes. The overexpression of TcLEC2 in cacao explants greatly increased the frequency of regeneration of stably transformed somatic embryos. TcLEC2 overexpressing cotyledon explants exhibited a very high level of embryogenic competency and when cultured on hormone free medium, exhibited an iterative embryogenic chain-reaction. Conclusions Our study revealed essential roles of TcLEC2 during both zygotic and somatic embryo development. Collectively, our evidence supports the conclusion that TcLEC2 is a functional ortholog of AtLEC2 and that it is involved in similar genetic regulatory networks during cacao somatic embryogenesis. To our knowledge, this is the first detailed report of the functional analysis of a LEC2 ortholog in a species other then Arabidopsis. TcLEC2 could potentially be used as a biomarker for the improvement of the SE process and screen for elite varieties in cacao germplasm. PMID:24758406

A continuous culture system of direct somatic embryogenesis in microspore-derived embryos of Brassica juncea.

PubMed

Prabhudesai, V; Bhaskaran, S

1993-03-01

An efficient culture system has been developed for repeated cycles of somatic embryogenesis in microspore-derived embryos of Brassica juncea without a callus phase. Haploid embryos produced through anther culture showed a high propensity for direct production of somatic embryos in response to 2 mgL(-1) BA and 0.1 mgL(-1) NAA. The embryogenic cultures which comprised the elongated embryonal axis of microspore-derived embryos when explanted and grown on the medium of same composition produced a large number of secondary embryos. These somatic embryos in turn underwent axis elongation and produced more somatic embryos when explanted and cultured. This cycle of repetitive somatic embryogenesis continued with undiminished vigour passage after passage and was monitored for more than a year. Somatic embryos from any passage when isolated at cotyledonary stage and grown on auxin-free medium for 5 days and then on a medium containing NAA (0.1 mgL(-1)), developed into complete plants with a profuse root system and were easily established in the soil. The cytology of the root tips of these plants confirmed their haploid nature. The total absence of callus phase makes the system ideal for continuous cloning of androgenic lines, Agrobacterium-mediated transformation and mutation induction studies.

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

PubMed

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

2016-11-05

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. Copyright © 2016. Published by Elsevier Ltd.

Somatic embryogenesis and plant regeneration in Carica papaya L. tissue culture derived from root explants.

PubMed

Chen, M H; Wang, P J; Maeda, E

1987-10-01

The regeneration potential of shoot tip, stem, leaf, cotyledon and root explants of two papaya cultivars (Carica papaya cv. 'Solo' and cv. 'Sunrise') were studed. Callus induction of these two cultivars of papaya showed that the shoot tips and stems are most suitable for forming callus, while leaves, cotyledons and roots are comparatively difficult to induce callus. Callus induction also varied with the varities. Somatic embryogenesis was obtained from 3-month-old root cultures. A medium containing half strength of MS inorganic salts, 160 mg/l adenine sulfate, 1.0 mg/1 NAA, 0.5 mg/1 kinetin and 1.0 mg/1 GA3 was optimal for embryogenesis. The callus maintained high regenerative capacity after two years of culture on this medium. Plants derived from somatic embryos were obtained under green-house conditions.

Enhanced somatic embryogenesis in Theobroma cacao using the homologous BABY BOOM transcription factor.

PubMed

Florez, Sergio L; Erwin, Rachel L; Maximova, Siela N; Guiltinan, Mark J; Curtis, Wayne R

2015-05-16

Theobroma cacao, the chocolate tree, is an important economic crop in East Africa, South East Asia, and South and Central America. Propagation of elite varieties has been achieved through somatic embryogenesis (SE) but low efficiencies and genotype dependence still presents a significant limitation for its propagation at commercial scales. Manipulation of transcription factors has been used to enhance the formation of SEs in several other plant species. This work describes the use of the transcription factor Baby Boom (BBM) to promote the transition of somatic cacao cells from the vegetative to embryonic state. An ortholog of the Arabidopsis thaliana BBM gene (AtBBM) was characterized in T. cacao (TcBBM). TcBBM expression was observed throughout embryo development and was expressed at higher levels during SE as compared to zygotic embryogenesis (ZE). TcBBM overexpression in A. thaliana and T. cacao led to phenotypes associated with SE that did not require exogenous hormones. While transient ectopic expression of TcBBM provided only moderate enhancements in embryogenic potential, constitutive overexpression dramatically increased SE proliferation but also appeared to inhibit subsequent development. Our work provides validation that TcBBM is an ortholog to AtBBM and has a specific role in both somatic and zygotic embryogenesis. Furthermore, our studies revealed that TcBBM transcript levels could serve as a biomarker for embryogenesis in cacao tissue. Results from transient expression of TcBBM provide confirmation that transcription factors can be used to enhance SE without compromising plant development and avoiding GMO plant production. This strategy could compliment a hormone-based method of reprogramming somatic cells and lead to more precise manipulation of SE at the regulatory level of transcription factors. The technology would benefit the propagation of elite varieties with low regeneration potential as well as the production of transgenic plants, which similarly requires somatic cell reprogramming.

Differential proteome analysis during early somatic embryogenesis in Musa spp. AAA cv. Grand Naine.

PubMed

Kumaravel, Marimuthu; Uma, Subbaraya; Backiyarani, Suthanthiram; Saraswathi, Marimuthu Somasundaram; Vaganan, Muthu Mayil; Muthusamy, Muthusamy; Sajith, Kallu Purayil

2017-01-01

Endogenous hormone secretion proteins along with stress and defense proteins play predominant role in banana embryogenesis. This study reveals the underlying molecular mechanism during transition from vegetative to embryogenic state. Banana (Musa spp.) is well known globally as a food fruit crop for millions. The requirement of quality planting material of banana is enormous. Although mass multiplication through tissue culture is in vogue, high-throughput techniques like somatic embryogenesis (SE) as a mass multiplication tool needs to be improved. Apart from clonal propagation, SE has extensive applications in genetic improvement and mutation. SE in banana is completely genome-dependent and most of the commercial cultivars exhibit recalcitrance. Thus, understanding the molecular basis of embryogenesis in Musa will help to develop strategies for mass production of quality planting material. In this study, differentially expressed proteins between embryogenic calli (EC) and non-embryogenic calli (NEC) with respect to the explant, immature male flower buds (IMFB), of cv. Grand Naine (AAA) were determined using two-dimensional gel electrophoresis (2DE). The 2DE results were validated through qRT-PCR. In total, 65 proteins were identified: 42 were highly expressed and 23 were less expressed in EC compared to NEC and IMFB. qRT-PCR analysis of five candidate proteins, upregulated in EC, were well correlated with expression at transcript level. Further analysis of proteins showed that embryogenesis in banana is associated with the control of oxidative stress. The regulation of ROS scavenging system and protection of protein structure occurred in the presence of heat shock proteins. Alongside, high accumulation of stress-related cationic peroxidase and plant growth hormone-related proteins like indole-3-pyruvate monooxygenase and adenylate isopentenyltransferase in EC revealed the association with the induction of SE.

Mps1 (Monopolar Spindle 1) Protein Inhibition Affects Cellular Growth and Pro-Embryogenic Masses Morphology in Embryogenic Cultures of Araucaria angustifolia (Araucariaceae).

PubMed

Douétts-Peres, Jackellinne C; Cruz, Marco Antônio L; Reis, Ricardo S; Heringer, Angelo S; de Oliveira, Eduardo A G; Elbl, Paula M; Floh, Eny I S; Silveira, Vanildo; Santa-Catarina, Claudete

2016-01-01

Somatic embryogenesis has been shown to be an efficient tool for studying processes based on cell growth and development. The fine regulation of the cell cycle is essential for proper embryo formation during the process of somatic embryogenesis. The aims of the present work were to identify and perform a structural and functional characterization of Mps1 and to analyze the effects of the inhibition of this protein on cellular growth and pro-embryogenic mass (PEM) morphology in embryogenic cultures of A. angustifolia. A single-copy Mps1 gene named AaMps1 was retrieved from the A. angustifolia transcriptome database, and through a mass spectrometry approach, AaMps1 was identified and quantified in embryogenic cultures. The Mps1 inhibitor SP600125 (10 μM) inhibited cellular growth and changed PEMs, and these effects were accompanied by a reduction in AaMps1 protein levels in embryogenic cultures. Our work has identified the Mps1 protein in a gymnosperm species for the first time, and we have shown that inhibiting Mps1 affects cellular growth and PEM differentiation during A. angustifolia somatic embryogenesis. These data will be useful for better understanding cell cycle control during somatic embryogenesis in plants.

Mps1 (Monopolar Spindle 1) Protein Inhibition Affects Cellular Growth and Pro-Embryogenic Masses Morphology in Embryogenic Cultures of Araucaria angustifolia (Araucariaceae)

PubMed Central

Douétts-Peres, Jackellinne C.; Cruz, Marco Antônio L.; Reis, Ricardo S.; Heringer, Angelo S.; de Oliveira, Eduardo A. G.; Elbl, Paula M.; Floh, Eny I. S.; Silveira, Vanildo

2016-01-01

Somatic embryogenesis has been shown to be an efficient tool for studying processes based on cell growth and development. The fine regulation of the cell cycle is essential for proper embryo formation during the process of somatic embryogenesis. The aims of the present work were to identify and perform a structural and functional characterization of Mps1 and to analyze the effects of the inhibition of this protein on cellular growth and pro-embryogenic mass (PEM) morphology in embryogenic cultures of A. angustifolia. A single-copy Mps1 gene named AaMps1 was retrieved from the A. angustifolia transcriptome database, and through a mass spectrometry approach, AaMps1 was identified and quantified in embryogenic cultures. The Mps1 inhibitor SP600125 (10 μM) inhibited cellular growth and changed PEMs, and these effects were accompanied by a reduction in AaMps1 protein levels in embryogenic cultures. Our work has identified the Mps1 protein in a gymnosperm species for the first time, and we have shown that inhibiting Mps1 affects cellular growth and PEM differentiation during A. angustifolia somatic embryogenesis. These data will be useful for better understanding cell cycle control during somatic embryogenesis in plants. PMID:27064899

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.

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.

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. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Identification and characterization of bZIP-type transcription factors involved in carrot (Daucus carota L.) somatic embryogenesis.

PubMed

Guan, Yucheng; Ren, Haibo; Xie, He; Ma, Zeyang; Chen, Fan

2009-10-01

Seed dormancy is an important adaptive trait that enables seeds of many species to remain quiescent until conditions become favorable for germination. Abscisic acid (ABA) plays an important role in these developmental processes. Like dormancy and germination, the elongation of carrot somatic embryo radicles is retarded by sucrose concentrations at or above 6%, and normal growth resumes at sucrose concentrations below 3%. Using a yeast one-hybrid screening system, we isolated two bZIP-type transcription factors, CAREB1 and CAREB2, from a cDNA library prepared from carrot somatic embryos cultured in a high-sucrose medium. Both CAREB1 and CAREB2 were localized to the nucleus, and specifically bound to the ABA response element (ABRE) in the Dc3 promoter. Expression of CAREB2 was induced in seedlings by drought and exogenous ABA application; whereas expression of CAREB1 increased during late embryogenesis, and reduced dramatically when somatic embryos were treated with fluridone, an inhibitor of ABA synthesis. Overexpression of CAREB1 caused somatic embryos to develop slowly when cultured in low-sucrose medium, and retarded the elongation of the radicles. These results indicate that CAREB1 and CAREB2 have similar DNA-binding activities, but play different roles during carrot development. Our results indicate that CAREB1 functions as an important trans-acting factor in the ABA signal transduction pathway during carrot somatic embryogenesis.

Somatic embryogenesis in Hedychium bousigonianum

USDA-ARS?s Scientific Manuscript database

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

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.

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.

Morphological analyses and variation in carbohydrate content during the maturation of somatic embryos of Carica papaya.

PubMed

Vale, Ellen Moura; Reis, Ricardo Souza; Passamani, Lucas Zanchetta; Santa-Catarina, Claudete; Silveira, Vanildo

2018-03-01

Efficient protocols for somatic embryogenesis of papaya ( Carica papaya L.) have great potential for selecting elite hybrid genotypes. Addition of polyethylene glycol (PEG), a nonplasmolyzing osmotic agent, to a maturation medium increases the production of somatic embryos in C . papaya . To study the effects of PEG on somatic embryogenesis of C . papaya , we analyzed somatic embryo development and carbohydrate profile changes during maturation treatments with PEG (6%) or without PEG (control). PEG treatment (6%) increased the number of normal mature somatic embryos followed by somatic plantlet production. In both control and PEG treatments, pro-embryogenic differentiation to the cotyledonary stage was observed and was significantly higher with PEG treatment. Histomorphological analysis of embryonic cultures with PEG revealed meristematic centers containing small isodiametric cells with dense cytoplasm and evident nuclei. Concomitant with the increase in the differentiation of somatic embryos in PEG cultures, there was an increase in the endogenous content of sucrose and starch, which appears to be related to a rising demand for energy, a key point in the conversion of C . papaya somatic embryos. The endogenous carbohydrate profile may be a valuable parameter for developing optimized protocols for the maturation of somatic embryos in papaya.

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

PubMed Central

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

2015-01-01

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

Spatio-Temporal Accumulation and Activity of Calcium-Dependent Protein Kinases during Embryogenesis, Seed Development, and Germination in Sandalwood1

PubMed Central

Anil, Veena S.; Harmon, Alice C.; Rao, K. Sankara

2000-01-01

Western-blot analysis and protein kinase assays identified two Ca2+-dependent protein kinases (CDPKs) of 55 to 60 kD in soluble protein extracts of embryogenic cultures of sandalwood (Santalum album L.). However, these sandalwood CDPKs (swCDPKs) were absent in plantlets regenerated from somatic embryos. swCDPKs exhibited differential expression (monitored at the level of the protein) and activity in different developmental stages. Zygotic embryos, seedlings, and endosperm showed high accumulation of swCDPK, but the enzyme was not detected in the soluble proteins of shoots and flowers. swCDPK exhibited a temporal pattern of expression in endosperm, showing high accumulation and activity in mature fruit and germinating stages; the enzyme was localized strongly in the storage bodies of the endosperm cells. The study also reports for the first time to our knowledge a post-translational inhibition/inactivation of swCDPK in zygotic embryos during seed dormancy and early stages of germination. The temporal expression of swCDPK during somatic/zygotic embryogenesis, seed maturation, and germination suggests involvement of the enzyme in these developmental processes. PMID:10759499

Spatio-temporal accumulation and activity of calcium-dependent protein kinases during embryogenesis, seed development, and germination in sandalwood.

PubMed

Anil, V S; Harmon, A C; Rao, K S

2000-04-01

Western-blot analysis and protein kinase assays identified two Ca(2+)-dependent protein kinases (CDPKs) of 55 to 60 kD in soluble protein extracts of embryogenic cultures of sandalwood (Santalum album L.). However, these sandalwood CDPKs (swCDPKs) were absent in plantlets regenerated from somatic embryos. swCDPKs exhibited differential expression (monitored at the level of the protein) and activity in different developmental stages. Zygotic embryos, seedlings, and endosperm showed high accumulation of swCDPK, but the enzyme was not detected in the soluble proteins of shoots and flowers. swCDPK exhibited a temporal pattern of expression in endosperm, showing high accumulation and activity in mature fruit and germinating stages; the enzyme was localized strongly in the storage bodies of the endosperm cells. The study also reports for the first time to our knowledge a post-translational inhibition/inactivation of swCDPK in zygotic embryos during seed dormancy and early stages of germination. The temporal expression of swCDPK during somatic/zygotic embryogenesis, seed maturation, and germination suggests involvement of the enzyme in these developmental processes.

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

PubMed

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

2013-09-01

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

Effects of GhWUS from upland cotton (Gossypium hirsutum L.) on somatic embryogenesis and shoot regeneration.

PubMed

Xiao, Yanqing; Chen, Yanli; Ding, Yanpeng; Wu, Jie; Wang, Peng; Yu, Ya; Wei, Xi; Wang, Ye; Zhang, Chaojun; Li, Fuguang; Ge, Xiaoyang

2018-05-01

The WUSCHEL (WUS) gene encodes a plant-specific homeodomain-containing transcriptional regulator, which plays important roles during embryogenesis, as well as in the formation of shoot and flower meristems. Here, we isolated two homologues of Arabidopsis thaliana WUS (AtWUS), GhWUS1a_At and GhWUS1b_At, from upland cotton (Gossypium hirsutum). Domain analysis suggested that the two putative GhWUS proteins contained a highly conserved DNA-binding HOX domain and a WUS-box. Expression profile analysis showed that GhWUSs were predominantly expressed during the embryoid stage. Ectopic expression of GhWUSs in Arabidopsis could induce somatic embryo and shoot formation from seedling root tips. Furthermore, in the absence of exogenous hormone, overexpression of GhWUSs in Arabidopsis could promote shoot regeneration from excised roots, and in the presence of exogenous auxin, excised roots expressing GhWUS could be induced to produce somatic embryo. In addition, expression of the chimeric GhWUS repressor in cotton callus inhibited embryogenic callus formation. Our results show that GhWUS is an important regulator of somatic embryogenesis and shoot regeneration. Copyright © 2018 Elsevier B.V. All rights reserved.

Comparison of somatic embryogenesis in Medicago sativa and Medicago truncatula.

PubMed

Hoori, F; Ehsanpour, A A; Mostajeran, A

2007-02-01

In this study, the regeneration through embryogenesis of two species of Medicago were studied. Seeds of Medicago sativa cv. Rehnani and M. truncatula line A17 were grown on MS medium. After 4-6 weeks, segments of leaf and stem from two species were transferred to MS medium containing 2 mg L(-1) NAA, 2,4-D and Kinetin. The results indicated that callus formation from leaf explants of M. sativa was higher than M. trancatula. In the next stage, media with different combinations of auxin, cytokinin or ethinyl estradiol were provided for regeneration. Then in two stages, explants of leaf and stem of two species were transferred on these media. Results after 3-6 weeks showed that in medium containing NAA and TDZ, stem pieces ofM. sativa produced shoots while leaf pieces on NAA and ethinyl estradiol formed roots. Leaf explants of M. truncatula in the medium containing NAA and BAP, produced somatic embryos. Also in media with auxin and ethinyl estradiol, somatic embryos were formed on calli of two species. Ethinyl estradiol and auxin together can induce somatic embryogenesis and root production on calli and stem or leaf explants.

Efficient plant regeneration through somatic embryogenesis from callus cultures of Oncidium (Orchidaceae).

PubMed

Chen, J -T.; Chang, W -C.

2000-12-07

An efficient method was established for high frequency somatic embryogenesis and plant regeneration from callus cultures of a hybrid of sympodial orchid (Oncidium 'Gower Ramsey'). Compact and yellow-white embryogenic calli formed from root tips and cut ends of stem and leaf segments on 1/2 MS [11] basal medium supplemented with 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ, 0.1-3 mg/l), 2,4-dichlorophenoxyacetic acid (2,4-D, 3-10 mg/l) and peptone (1 g/l) for 4-7 weeks. Embryogenic callus was maintained by subculture on the same medium for callus induction and proliferated 2-4 times (fresh weight) in 1 month. Initiation of somatic embryogenesis and development up to the protocorm-like-bodies (PLBs) from callus cultures was achieved on hormone-free basal medium. Regenerants were recovered from somatic embryos (SEs) after transfer to the same medium and showed normal development. The optimized protocol required about 12-14 weeks from the initiation of callus to the plantlet formation. Generally, the frequency of embryo formation of root-derived callus was higher than stem- and leaf-derived calli. Combinations of naphthaleneacetic acid (NAA) and TDZ significantly promoted embryo formation from callus cultures. The high-frequency (93.8%) somatic embryogenesis and an average of 29.1 SEs per callus (3x3 mm(2)) was found in root-derived callus on a basal medium supplemented with 0.1 mg/l NAA and 3 mg/l TDZ. Almost all the SEs converted and the plantlets grew well with an almost 100% survival rate when potted in sphagnum moss and acclimatized in the greenhouse.

Gene expression patterns during somatic embryo development and germination in maize Hi II callus cultures.

PubMed

Che, Ping; Love, Tanzy M; Frame, Bronwyn R; Wang, Kan; Carriquiry, Alicia L; Howell, Stephen H

2006-09-01

Gene expression patterns were profiled during somatic embryogenesis in a regeneration-proficient maize hybrid line, Hi II, in an effort to identify genes that might be used as developmental markers or targets to optimize regeneration steps for recovering maize plants from tissue culture. Gene expression profiles were generated from embryogenic calli induced to undergo embryo maturation and germination. Over 1,000 genes in the 12,060 element arrays showed significant time variation during somatic embryo development. A substantial number of genes were downregulated during embryo maturation, largely histone and ribosomal protein genes, which may result from a slowdown in cell proliferation and growth during embryo maturation. The expression of these genes dramatically recovered at germination. Other genes up-regulated during embryo maturation included genes encoding hydrolytic enzymes (nucleases, glucosidases and proteases) and a few storage genes (an alpha-zein and caleosin), which are good candidates for developmental marker genes. Germination is accompanied by the up-regulation of a number of stress response and membrane transporter genes, and, as expected, greening is associated with the up-regulation of many genes encoding photosynthetic and chloroplast components. Thus, some, but not all genes typically associated with zygotic embryogenesis are significantly up or down-regulated during somatic embryogenesis in Hi II maize line regeneration. Although many genes varied in expression throughout somatic embryo development in this study, no statistically significant gene expression changes were detected between total embryogenic callus and callus enriched for transition stage somatic embryos.

Evaluation of haemoglobin (erythrogen): for improved somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L. cv. SVPR 2).

PubMed

Ganesan, M; Jayabalan, N

2004-10-01

Somatic embryogenesis in cotton (Gossypium hirsutum L.) is accelerated when the plant regeneration medium is supplemented with haemoglobin (erythrogen). In cotton SVPR 2 lines, a higher frequency of embryoid formation was observed when the medium contained 400 mg/l haemoglobin. Fresh weight of the callus, rate of embryoid induction, number of embryoids formed and the percentage of plant regeneration from somatic embryos were increased. Among the two different cultivars tested, MCU 11 showed no response to the presence of haemoglobin when compared to SVPR 2, and embryogenic callus formation was completely absent in the former. Medium containing MS salts, 100 mg/l myo-inositol , 0.3 mg/l thiamine-HCL, 0.3 mg/l Picloram (PIC), 0.1 mg/l kinetin and 400 mg/l haemoglobin effected a better response with respect to embryogenic callus induction. After 8 weeks of culture, a high frequency of embryoid induction was observed on medium containing MS basal salts, 100 mg/l myo-inositol, 0.3 mg/l PIC , 0.1 mg/l isopentenyl adenine, 1.0 g/l NH4NO3 and 400 mg/l haemoglobin. Plant regeneration was observed in 75.8% of the mature somatic embryos, and whole plant regeneration was achieved within 6-7 months of culture. The regenerated plantlets were fertile and similar to in vivo-grown, seed-derived plants except that they were phenotypically smaller. A positive influence of haemoglobin was observed at concentrations up to 400 mg/l at all stages of somatic embryogenesis. The increase in the levels of antioxidant enzyme activities, for example superoxide dismutase and peroxidase, indicated the presence of excess oxygen uptake and the stressed condition of the plant tissues that arose from haemoglobin supplementation. This increased oxygen uptake and haemoglobin-mediated stress appeared to accelerate somatic embryogenesis in cotton.

Annotation of Differentially Expressed Genes in the Somatic Embryogenesis of Musa and Their Location in the Banana Genome

PubMed Central

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. PMID:24027442

Truncation of LEAFY COTYLEDON1 Protein Is Required for Asexual Reproduction in Kalanchoë daigremontiana1[OPEN

PubMed Central

Garcês, Helena M.P.; Koenig, Daniel; Townsley, Brad T.; Kim, Minsung; Sinha, Neelima R.

2014-01-01

Kalanchoë daigremontiana reproduces asexually by generating numerous plantlets on its leaf margins. The formation of plantlets requires the somatic initiation of organogenic and embryogenic developmental programs in the leaves. However, unlike normal embryogenesis in seeds, leaf somatic embryogenesis bypasses seed dormancy to form viable plantlets. In Arabidopsis (Arabidopsis thaliana), seed dormancy and embryogenesis are initiated by the transcription factor LEAFY COTYLEDON1 (LEC1). The K. daigremontiana ortholog of LEC1 is expressed during leaf somatic embryo development. However, KdLEC1 encodes for a LEC1-type protein that has a unique B domain, with 11 unique amino acids and a premature stop codon. Moreover, the truncated KdLEC1 protein is not functional in Arabidopsis. Here, we show that K. daigremontiana transgenic plants expressing a functional, chimeric KdLEC1 gene under the control of Arabidopsis LEC1 promoter caused several developmental defects to leaf somatic embryos, including seed dormancy characteristics. The dormant plantlets also behaved as typical dormant seeds. Transgenic plantlets accumulated oil bodies and responded to the abscisic acid biosynthesis inhibitor fluridone, which broke somatic-embryo dormancy and promoted their normal development. Our results indicate that having a mutated form of LEC1 gene in K. daigremontiana is essential to bypass dormancy in the leaf embryos and generate viable plantlets, suggesting that the loss of a functional LEC1 promotes viviparous leaf somatic embryos and thus enhances vegetative propagation in K. daigremontiana. Mutations resulting in truncated LEC1 proteins may have been of a selective advantage in creating somatic propagules, because such mutations occurred independently in several Kalanchoë species, which form plantlets constitutively. PMID:24664206

Truncation of LEAFY COTYLEDON1 protein is required for asexual reproduction in Kalanchoë daigremontiana.

PubMed

Garcês, Helena M P; Koenig, Daniel; Townsley, Brad T; Kim, Minsung; Sinha, Neelima R

2014-05-01

Kalanchoë daigremontiana reproduces asexually by generating numerous plantlets on its leaf margins. The formation of plantlets requires the somatic initiation of organogenic and embryogenic developmental programs in the leaves. However, unlike normal embryogenesis in seeds, leaf somatic embryogenesis bypasses seed dormancy to form viable plantlets. In Arabidopsis (Arabidopsis thaliana), seed dormancy and embryogenesis are initiated by the transcription factor LEAFY COTYLEDON1 (LEC1). The K. daigremontiana ortholog of LEC1 is expressed during leaf somatic embryo development. However, KdLEC1 encodes for a LEC1-type protein that has a unique B domain, with 11 unique amino acids and a premature stop codon. Moreover, the truncated KdLEC1 protein is not functional in Arabidopsis. Here, we show that K. daigremontiana transgenic plants expressing a functional, chimeric KdLEC1 gene under the control of Arabidopsis LEC1 promoter caused several developmental defects to leaf somatic embryos, including seed dormancy characteristics. The dormant plantlets also behaved as typical dormant seeds. Transgenic plantlets accumulated oil bodies and responded to the abscisic acid biosynthesis inhibitor fluridone, which broke somatic-embryo dormancy and promoted their normal development. Our results indicate that having a mutated form of LEC1 gene in K. daigremontiana is essential to bypass dormancy in the leaf embryos and generate viable plantlets, suggesting that the loss of a functional LEC1 promotes viviparous leaf somatic embryos and thus enhances vegetative propagation in K. daigremontiana. Mutations resulting in truncated LEC1 proteins may have been of a selective advantage in creating somatic propagules, because such mutations occurred independently in several Kalanchoë species, which form plantlets constitutively.

Proteome profiling of early seed development in Cunninghamia lanceolata (Lamb.) Hook

PubMed Central

Shi, Jisen; Zhen, Yan; Zheng, Ren-Hua

2010-01-01

Knowledge of the proteome of the early gymnosperm embryo could provide important information for optimizing plant cloning procedures and for establishing platforms for research into plant development/regulation and in vitro transgenic studies. Compared with angiosperms, it is more difficult to induce somatic embryogenesis in gymnosperms; success in this endeavour could be increased, however, if proteomic information was available on the complex, dynamic, and multistage processes of gymnosperm embryogenesis in vivo. A proteomic analysis of Chinese fir seeds in six developmental stages was carried out during early embryogenesis. Proteins were extracted from seeds dissected from immature cones and separated by two-dimensional difference gel electrophoresis. Analysis with DeCyder 6.5 software revealed 136 spots that differed in kinetics of appearance. Analysis by liquid chromatography coupled to tandem mass spectrometry and MALDI-TOF mass spectrometry identified proteins represented by 71 of the spots. Functional annotation of these seed proteins revealed their involvement in programmed cell death and chromatin modification, indicating that the proteins may play a central role in determining the number of zygotic embryos generated and controlling embryo patterning and shape remodelling. The analysis also revealed other proteins involved in carbon metabolism, methionine metabolism, energy production, protein storage, synthesis and stabilization, disease/defence, the cytoskeleton, and embryo development. The comprehensive protein expression profiles generated by our study provide new insights into the complex developmental processes in the seeds of the Chinese fir. PMID:20363864

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.

A Small RNA-Catalytic Argonaute Pathway Tunes Germline Transcript Levels to Ensure Embryonic Divisions

PubMed Central

Gerson-Gurwitz, Adina; Wang, Shaohe; Sathe, Shashank; Green, Rebecca; Yeo, Gene W.; Oegema, Karen; Desai, Arshad

2016-01-01

SUMMARY Multiple division cycles without growth are a characteristic feature of early embryogenesis. The female germline loads proteins and RNAs into oocytes to support these divisions, which lack many quality control mechanisms operating in somatic cells undergoing growth. Here we describe a small RNA-Argonaute pathway that ensures early embryonic divisions in C. elegans by employing catalytic slicing activity to broadly tune, instead of silence, germline gene expression. Misregulation of one target, a kinesin-13 microtubule depolymerase, underlies a major phenotype associated with pathway loss. Tuning of target transcript levels is guided by density of homologous small RNAs, whose generation must ultimately be related to target sequence. Thus, the tuning action of a small RNA-catalytic Argonaute pathway generates oocytes capable of supporting embryogenesis. We speculate that the specialized nature of germline chromatin led to emergence of small RNA-catalytic Argonaute pathways in the female germline as a post-transcriptional control layer to optimize oocyte composition. PMID:27020753

Construction of a high-density linkage map and mapping quantitative trait loci for somatic embryogenesis using leaf petioles as explants in upland cotton (Gossypium hirsutum L.).

PubMed

Xu, Zhenzhen; Zhang, Chaojun; Ge, Xiaoyang; Wang, Ni; Zhou, Kehai; Yang, Xiaojie; Wu, Zhixia; Zhang, Xueyan; Liu, Chuanliang; Yang, Zuoren; Li, Changfeng; Liu, Kun; Yang, Zhaoen; Qian, Yuyuan; Li, Fuguang

2015-07-01

The first high-density linkage map was constructed to identify quantitative trait loci (QTLs) for somatic embryogenesis (SE) in cotton ( Gossypium hirsutum L.) using leaf petioles as explants. Cotton transformation is highly limited by only a few regenerable genotypes and the lack of understanding of the genetic and molecular basis of somatic embryogenesis (SE) in cotton (Gossypium hirsutum L.). To construct a more saturated linkage map and further identify quantitative trait loci (QTLs) for SE using leaf petioles as explants, a high embryogenesis frequency line (W10) from the commercial Chinese cotton cultivar CRI24 was crossed with TM-1, a genetic standard upland cotton with no embryogenesis frequency. The genetic map spanned 2300.41 cM in genetic distance and contained 411 polymorphic simple sequence repeat (SSR) loci. Of the 411 mapped loci, 25 were developed from unigenes identified for SE in our previous study. Six QTLs for SE were detected by composite interval mapping method, each explaining 6.88-37.07% of the phenotypic variance. Single marker analysis was also performed to verify the reliability of QTLs detection, and the SSR markers NAU3325 and DPL0209 were detected by the two methods. Further studies on the relatively stable and anchoring QTLs/markers for SE in an advanced population of W10 × TM-1 and other cross combinations with different SE abilities may shed light on the genetic and molecular mechanism of SE in cotton.

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

PubMed Central

2011-01-01

Background Hydroxyproline rich glycoproteins (HRGPs) are implicated to have a role in many aspects of plant growth and development but there is limited knowledge about their localization and function during somatic embryogenesis of higher plants. In this study, the localization and function of hydroxyproline rich glycoproteins in embryogenic cells (ECs) and somatic embryos of banana were investigated by using immunobloting and immunocytochemistry with monoclonal JIM11 and JIM20 antibodies as well as by treatment with 3,4-dehydro-L-proline (3,4-DHP, an inhibitor of extensin biosynthesis), and by immunomodulation with the JIM11 antibody. Results Immunofluorescence labelling of JIM11 and JIM20 hydroxyproline rich glycoprotein epitopes was relatively weak in non-embryogenic cells (NECs), mainly on the edge of small cell aggregates. On the other hand, hydroxyproline rich glycoprotein epitopes were found to be enriched in early embryogenic cells as well as in various developmental stages of somatic embryos. Embryogenic cells (ECs), proembryos and globular embryos showed strong labelling of hydroxyproline rich glycoprotein epitopes, especially in their cell walls and outer surface layer, so-called extracellular matrix (ECM). This hydroxyproline rich glycoprotein signal at embryo surfaces decreased and/or fully disappeared during later developmental stages (e.g. pear-shaped and cotyledonary stages) of embryos. In these later developmental embryogenic stages, however, new prominent hydroxyproline rich glycoprotein labelling appeared in tri-cellular junctions among parenchymatic cells inside these embryos. Overall immunofluorescence labelling of late stage embryos with JIM20 antibody was weaker than that of JIM11. Western blot analysis supported the above immunolocalization data. The treatment with 3,4-DHP inhibited the development of embryogenic cells and decreased the rate of embryo germination. Embryo-like structures, which developed after 3,4-DHP treatment showed aberrant non-compact epidermis with discontinuous ECM at the outer surface as well as much less immunolabelling with the JIM11 antibody. This treatment also decreased the plant regeneration capacity in embryogenic banana cultures. Finally, immunomodulation of surface hydroxyproline rich glycoproteins by co-culture of embryos with the JIM11 antibody resulted in a much lower germination capacity of these embryos. Conclusions These results suggest that hydroxyproline rich glycoproteins play an important developmental role, especially in the process of regeneration and germination of embryos during plant regeneration via somatic embryogenesis. Proper content and localization of hydroxyproline rich glycoproteins seem to be essential for the formation and regeneration of banana somatic embryos. PMID:21349190

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

PubMed

Maximova, Siela N; Florez, Sergio; Shen, Xiangling; Niemenak, Nicolas; Zhang, Yufan; Curtis, Wayne; Guiltinan, Mark J

2014-07-16

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. 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. 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. These differences in gene transcript levels associated with critical processes during seed development are consistent with the fact that somatic embryos do not fully develop the large storage cotyledons found in zygotic embryos. These results provide insight towards design of improved protocols for cacao somatic embryogenesis.

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. These differences in gene transcript levels associated with critical processes during seed development are consistent with the fact that somatic embryos do not fully develop the large storage cotyledons found in zygotic embryos. These results provide insight towards design of improved protocols for cacao somatic embryogenesis. PMID:25030026

Somatic embryogenesis in forestry: A practical approach to cloning the best trees

Treesearch

Alex M. Diner

1999-01-01

Trees as well as humans have two basic cell types based on genetic content: somatic cells and gametic or reproductive cells. Somatic cells, such as skin cells or the sapwood cells in a tree, have at least twice (2n) the base set of chromosomes. The reproductive cells (gametic cells) have a single (n) set of chromosomes.

Proteomic analysis of stipe explants reveals differentially expressed proteins involved in early direct somatic embryogenesis of the tree fern Cyathea delgadii Sternb.

PubMed

Domżalska, Lucyna; Kędracka-Krok, Sylwia; Jankowska, Urszula; Grzyb, Małgorzata; Sobczak, Mirosław; Rybczyński, Jan J; Mikuła, Anna

2017-05-01

Using cyto-morphological analysis of somatic embryogenesis (SE) in the tree fern Cyathea delgadii as a guide, we performed a comparative proteomic analysis in stipe explants undergoing direct SE. Plant material was cultured on hormone-free medium supplemented with 2% sucrose. Phenol extracted proteins were separated using two-dimensional gel electrophoresis (2-DE) and mass spectrometry was performed for protein identification. A total number of 114 differentially regulated proteins was identified during early SE, i.e. when the first cell divisions started and several-cell pro-embryos were formed. Proteins were assigned to seven functional categories: carbohydrate metabolism, protein metabolism, cell organization, defense and stress responses, amino acid metabolism, purine metabolism, and fatty acid metabolism. Carbohydrate and protein metabolism were found to be the most sensitive SE functions with the greatest number of alterations in the intensity of spots in gel. Differences, especially in non-enzymatic and structural protein abundance, are indicative for cell organization, including cytoskeleton rearrangement and changes in cell wall components. The highest induced changes concern those enzymes related to fatty acid metabolism. Global analysis of the proteome reveals several proteins that can represent markers for the first 16days of SE induction and expression in fern. The findings of this research improve the understanding of molecular processes involved in direct SE in C. delgadii. Copyright © 2017 Elsevier B.V. All rights reserved.

PRC2 Represses Hormone-Induced Somatic Embryogenesis in Vegetative Tissue of Arabidopsis thaliana

PubMed Central

Mozgová, Iva

2017-01-01

Many plant cells can be reprogrammed into a pluripotent state that allows ectopic organ development. Inducing totipotent states to stimulate somatic embryo (SE) development is, however, challenging due to insufficient understanding of molecular barriers that prevent somatic cell dedifferentiation. Here we show that Polycomb repressive complex 2 (PRC2)-activity imposes a barrier to hormone-mediated transcriptional reprogramming towards somatic embryogenesis in vegetative tissue of Arabidopsis thaliana. We identify factors that enable SE development in PRC2-depleted shoot and root tissue and demonstrate that the establishment of embryogenic potential is marked by ectopic co-activation of crucial developmental regulators that specify shoot, root and embryo identity. Using inducible activation of PRC2 in PRC2-depleted cells, we demonstrate that transient reduction of PRC2 activity is sufficient for SE formation. We suggest that modulation of PRC2 activity in plant vegetative tissue combined with targeted activation of developmental pathways will open possibilities for novel approaches to cell reprogramming. PMID:28095419

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.

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.

Glutathione and abscisic acid supplementation influences somatic embryo maturation and hormone endogenous levels during somatic embryogenesis in Podocarpus lambertii Klotzsch ex Endl.

PubMed

Fraga, Hugo Pacheco de Freitas; Vieira, Leila do Nascimento; Puttkammer, Catarina Corrêa; Dos Santos, Henrique Pessoa; Garighan, Julio de Andrade; Guerra, Miguel Pedro

2016-12-01

Here we propose a protocol for embryogenic cultures induction, proliferation and maturation for the Brazilian conifer Podocarpus lambertii, and investigated the effect of abscisic acid (ABA) and glutathione (GSH) supplementation on the maturation phase. ABA, zeatin (Z) and salicylic acid (SA) endogenous levels were quantified. Number of somatic embryos obtained in ABA-supplemented treatment was significant higher than in ABA-free treatment, showing the relevance of ABA supplementation during somatic embryos maturation. Histological analysis showed the stereotyped sequence of developmental stages in conifer somatic embryos, reaching the late torpedo-staged embryo. GSH supplementation in maturation culture medium improved the somatic embryos number and morphological features. GSH 0mM and GSH 0.1mM treatments correlated with a decreased ABA endogenous level during maturation, while GSH 0.5mM treatment showed constant levels. All treatments resulted in decreased Z endogenous levels, supporting the concept that cytokinins are important during the initial cell division but not for the later stages of embryo development. The lowest SA levels found in GSH 0.5mM treatment were coincident with early embryonic development, and this treatment resulted in the highest development of somatic embryos. Thus, a correlation between lower SA levels and improved somatic embryo formation can be hypothesized. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Somatic hybridization in Citrus: navel orange (C. sinensis Osb.) and grapefruit (C. paradisi Macf.).

PubMed

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

1989-11-01

Protoplasts of navel orange, isolated from embryogenic nucellar cell suspension culture, were fused with protoplasts of grapefruit isolated from leaf tissue. The fusion products were cultured in the hormone-free medium containing 0.6 M sucrose. Under the culture conditions, somatic embryogenesis of navel orange protoplasts was suppressed, while cell division of grapefruit mesophyll protoplasts was not induced. Six embryoids were obtained and three lines regenerated to complete plants through embryogenesis. Two of the regenerated lines exhibited intermediate morphological characteristics of the parents in the leaf shape. Chromosome counts showed that these regenerated plants had expected 36 chromosomes (2n=2x=18 for each parent). The rDNA analysis using biotin-labeled rRNA probes confirmed the presence of genomes from both parents in these plants. This somatic hybridization system would be useful for the practical Citrus breeding.

The pea aphid uses a version of the terminal system during oviparous, but not viviparous, development

PubMed Central

2013-01-01

Background In most species of aphid, female nymphs develop into either sexual or asexual adults depending on the length of the photoperiod to which their mothers were exposed. The progeny of these sexual and asexual females, in turn, develop in dramatically different ways. The fertilized oocytes of sexual females begin embryogenesis after being deposited on leaves (oviparous development) while the oocytes of asexual females complete embryogenesis within the mother (viviparous development). Compared with oviparous development, viviparous development involves a smaller transient oocyte surrounded by fewer somatic epithelial cells and a smaller early embryo that comprises fewer cells. To investigate whether patterning mechanisms differ between the earliest stages of the oviparous and viviparous modes of pea aphid development, we examined the expression of pea aphid orthologs of genes known to specify embryonic termini in other insects. Results Here we show that pea aphid oviparous ovaries express torso-like in somatic posterior follicle cells and activate ERK MAP kinase at the posterior of the oocyte. In addition to suggesting that some posterior features of the terminal system are evolutionarily conserved, our detection of activated ERK in the oocyte, rather than in the embryo, suggests that pea aphids may transduce the terminal signal using a mechanism distinct from the one used in Drosophila. In contrast with oviparous development, the pea aphid version of the terminal system does not appear to be used during viviparous development, since we did not detect expression of torso-like in the somatic epithelial cells that surround either the oocyte or the blastoderm embryo and we did not observe restricted activated ERK in the oocyte. Conclusions We suggest that while oviparous oocytes and embryos may specify posterior fate through an aphid terminal system, viviparous oocytes and embryos employ a different mechanism, perhaps one that does not rely on an interaction between the oocyte and surrounding somatic cells. Together, these observations provide a striking example of a difference in the fundamental events of early development that is both environmentally induced and encoded by the same genome. PMID:23552511

cDNA cloning, genomic organization and expression analysis during somatic embryogenesis of the translationally controlled tumor protein (TCTP) gene from Japanese larch (Larix leptolepis).

PubMed

Zhang, Li-Feng; Li, Wan-Feng; Han, Su-Ying; Yang, Wen-Hua; Qi, Li-Wang

2013-10-15

A full-length cDNA and genomic sequences of a translationally controlled tumor protein (TCTP) gene were isolated from Japanese larch (Larix leptolepis) and designated LaTCTP. The length of the cDNA was 1, 043 bp and contained a 504 bp open reading frame that encodes a predicted protein of 167 amino acids, characterized by two signature sequences of the TCTP protein family. Analysis of the LaTCTP gene structure indicated four introns and five exons, and it is the largest of all currently known TCTP genes in plants. The 5'-flanking promoter region of LaTCTP was cloned using an improved TAIL-PCR technique. In this region we identified many important potential cis-acting elements, such as a Box-W1 (fungal elicitor responsive element), a CAT-box (cis-acting regulatory element related to meristem expression), a CGTCA-motif (cis-acting regulatory element involved in MeJA-responsiveness), a GT1-motif (light responsive element), a Skn-1-motif (cis-acting regulatory element required for endosperm expression) and a TGA-element (auxin-responsive element), suggesting that expression of LaTCTP is highly regulated. Expression analysis demonstrated ubiquitous localization of LaTCTP mRNA in the roots, stems and needles, high mRNA levels in the embryonal-suspensor mass (ESM), browning embryogenic cultures and mature somatic embryos, and low levels of mRNA at day five during somatic embryogenesis. We suggest that LaTCTP might participate in the regulation of somatic embryo development. These results provide a theoretical basis for understanding the molecular regulatory mechanism of LaTCTP and lay the foundation for artificial regulation of somatic embryogenesis. © 2013.

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

PubMed Central

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

2012-01-01

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

Somatic embryogenesis and polyamines in woody plants

Treesearch

Rakesh Minocha; Subhash C. Minocha; Liisa Kaarina Simola

1995-01-01

The formation of whole plants from cultured cells is interesting not only because of its applications for mass propagation but also as a prime example of the process of controlled development and differentiation in plants. Cultures capable of producing somatic embryos with high frequency provide ideal experimental systems to study and understand the biochemical basis...

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

USDA-ARS?s Scientific Manuscript database

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

Somatic embryogenesis from flower explants of cocoa (Theobroma cacao L.).

PubMed

Silva, J J; Debergh, P

2001-01-01

Two types of flower explants, staminoides and petals, were used for in vitro induction of somatic embryos in cocoa. After 14 days in culture, we observed globular structures and callus formation on both types of explants. However, the better results were obtained on staminoides: 98.3% formed callus and 86.2% somatic embryos on Murashige and Skoog (1962) medium supplemented with sucrose, coconut water, 2,4-D, kinetin and agar.

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. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

The Phaseolus vulgaris PvTRX1h gene regulates plant hormone biosynthesis in embryogenic callus from common bean.

PubMed

Barraza, Aarón; Cabrera-Ponce, José L; Gamboa-Becerra, Roberto; Luna-Martínez, Francisco; Winkler, Robert; Álvarez-Venegas, Raúl

2015-01-01

Common bean is the most important grain legume in the human diet. Bean improvement efforts have been focused on classical breeding techniques because bean is recalcitrant to both somatic embryogenesis and in vitro regeneration. This study was undertaken to better understand the process of somatic embryogenesis in the common bean. We focused on the mechanisms by which somatic embryogenesis in plants is regulated and the interaction of these mechanisms with plant hormones. Specifically, we examined the role of the gene PvTRX1h, an ortholog of a major known histone lysine methyltransferase in plants, in somatic embryo generation. Given the problems with regeneration and transformation, we chose to develop and use regeneration-competent callus that could be successively transformed. Embryogenic calli of common bean were generated and transformed with the PvTRX1hRiA construction to down-regulate, by RNA interference, expression of the PvTRX1h gene. Plant hormone content was measured by mass spectrometry and gene expression was assessed by q-PCR. Detailed histological analysis was performed on selected transgenic embryogenic calli. It was determined that down-regulation of PvTRX1h gene was accompanied by altered concentrations of plant hormones in the calli. PvTRX1h regulated the expression of genes involved in auxin biosynthesis and embryogenic calli in which PvTRX1h was down-regulated were capable of differentiation into somatic embryos. Also, down-regulation of PvTRX1h showed increased transcript abundance of a gene coding for a second histone lysine methyltransferase, PvASHH2h. Accordingly, the PvTRX1h gene is involved in the synthesis of plant hormones in common bean callus. These results shed light on the crosstalk among histone methyltransferases and plant hormone signaling and on gene regulation during somatic embryo generation.

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

PubMed Central

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

2015-01-01

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

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

USDA-ARS?s Scientific Manuscript database

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...

Embryogenic competence acquisition in sugarcane callus is associated with differential H+ pump abundance and activity.

PubMed

Passamani, Lucas Z; Bertolazi, Amanda A; Ramos, Alessandro C; Santa-Catarina, Claudete; Thelen, Jay J; Silveira, Vanildo

2018-06-22

Somatic embryogenesis is an important biological process in several plant species, including sugarcane. Proteomics approaches have shown that H + pumps are differentially regulated during somatic embryogenesis; however, the relationship between H + flux and embryogenic competence is still unclear. This work aimed to elucidate the association between extracellular H + flux and somatic embryo maturation in sugarcane. We performed a microsomal proteomics analysis and analyzed changes in extracellular H + flux and H + pump (P-H + -ATPase, V-H + -ATPase and H + -PPase) activity in embryogenic and non-embryogenic callus. A total of 657 proteins were identified, 16 of which were H + pumps. We observed that P-H + -ATPase and H + -PPase were more abundant in embryogenic callus. Compared with non-embryogenic callus, embryogenic callus showed higher H + influx, especially on maturation day 14, as well as higher H+ pump activity (mainly P-H+-ATPase and H+-PPase activity). H+-PPase appears to be the major H + pump in embryogenic callus during somatic embryo formation, functioning in both vacuole acidification and PPi homeostasis. These results provide evidence for an association between higher H + pump protein abundance and, consequently, higher H + flux and embryogenic competence acquisition in the callus of sugarcane, allowing for optimization of the somatic embryo conversion process by modulating the activities of these H + pumps.

Animal cloning by somatic cell nuclear transfer.

PubMed

Smith, Lawrence C; Yoo, Jae-Gyu

2009-01-01

Animal cloning is becoming increasingly useful for its applications in biological inquiry and for its potential use in pharmaceutical, medical, and agricultural fields. Due to the complexity of the numerous steps required in reconstructing oocytes by nuclear transfer, detailed protocols are required to minimize the developmental damages inflicted during these manipulations and to standardize procedures across laboratories. Moreover, because oogenesis and early embryogenesis differ widely among mammalian species, it is essential that protocols be adapted according to each species concerned. Our objective here is to detail the protocols that have been most successful in producing laboratory and domestic animal clones.

Calcium-Mediated Signaling during Sandalwood Somatic Embryogenesis. Role for Exogenous Calcium as Second Messenger1

PubMed Central

Anil, Veena S.; Rao, K. Sankara

2000-01-01

The possible involvement of Ca2+-mediated signaling in the induction/regulation of somatic embryogenesis from pro-embryogenic cells of sandalwood (Santalum album) has been investigated. 45Ca2+-uptake studies and fura-2 fluorescence ratio photometry were used to measure changes in [Ca2+]cyt of pro-embryogenic cells in response to culture conditions conducive for embryo development. Sandalwood pro-embryogenic cell masses (PEMs) are obtained in the callus proliferation medium that contains the auxin 2,4-dichlorophenoxyacetic acid. Subculture of PEMs into the embryo differentiation medium, which lacks 2,4-dichlorophenoxyacetic acid and has higher osmoticum, results in a 4-fold higher 45Ca2+ incorporation into the symplast. Fura-2 ratiometric analysis corroboratively shows a 10- to 16-fold increase in the [Ca2+]cyt of PEMs, increasing from a resting concentration of 30 to 50 nm to 650 to 800 nm. Chelation of exogenous Ca2+ with ethyleneglycol-bis(aminoethyl ether)-N,N′-tetraacetic acid arrests such an elevation in [Ca2+]cyt. Exogenous Ca2+ when chelated or deprived also arrests embryo development and inhibits the accumulation of a sandalwood Ca2+-dependent protein kinase. However, such culture conditions do not cause cell death as the PEMs continue to proliferate to form larger cell clumps. Culture treatment with N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide reduced embryogenic frequency by 85%, indicating that blockage of Ca2+-mediated signaling pathway(s) involving sandalwood Ca2+-dependent protein kinase and/or calmodulin causes the inhibition of embryogenesis. The observations presented are evidence to suggest a second messenger role for exogenous Ca2+ during sandalwood somatic embryogenesis. PMID:10938349

Calcium-mediated signaling during sandalwood somatic embryogenesis. Role for exogenous calcium as second messenger.

PubMed

Anil, V S; Rao, K S

2000-08-01

The possible involvement of Ca(2+)-mediated signaling in the induction/regulation of somatic embryogenesis from pro-embryogenic cells of sandalwood (Santalum album) has been investigated. (45)Ca(2+)-uptake studies and fura-2 fluorescence ratio photometry were used to measure changes in [Ca(2+)](cyt) of pro-embryogenic cells in response to culture conditions conducive for embryo development. Sandalwood pro-embryogenic cell masses (PEMs) are obtained in the callus proliferation medium that contains the auxin 2,4-dichlorophenoxyacetic acid. Subculture of PEMs into the embryo differentiation medium, which lacks 2,4-dichlorophenoxyacetic acid and has higher osmoticum, results in a 4-fold higher (45)Ca(2+) incorporation into the symplast. Fura-2 ratiometric analysis corroboratively shows a 10- to 16-fold increase in the [Ca(2+)](cyt) of PEMs, increasing from a resting concentration of 30 to 50 nM to 650 to 800 nM. Chelation of exogenous Ca(2+) with ethyleneglycol-bis(aminoethyl ether)-N,N'-tetraacetic acid arrests such an elevation in [Ca(2+)](cyt). Exogenous Ca(2+) when chelated or deprived also arrests embryo development and inhibits the accumulation of a sandalwood Ca(2+)-dependent protein kinase. However, such culture conditions do not cause cell death as the PEMs continue to proliferate to form larger cell clumps. Culture treatment with N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide reduced embryogenic frequency by 85%, indicating that blockage of Ca(2+)-mediated signaling pathway(s) involving sandalwood Ca(2+)-dependent protein kinase and/or calmodulin causes the inhibition of embryogenesis. The observations presented are evidence to suggest a second messenger role for exogenous Ca(2+) during sandalwood somatic embryogenesis.

Disposable Bioreactors for Plant Micropropagation and Mass Plant Cell Culture

NASA Astrophysics Data System (ADS)

Ducos, Jean-Paul; Terrier, Bénédicte; Courtois, Didier

Different types of bioreactors are used at Nestlé R&D Centre - Tours for mass propagation of selected plant varieties by somatic embryogenesis and for large scale culture of plants cells to produce metabolites or recombinant proteins. Recent studies have been directed to cut down the production costs of these two processes by developing disposable cell culture systems. Vegetative propagation of elite plant varieties is achieved through somatic embryogenesis in liquid medium. A pilot scale process has recently been set up for the industrial propagation of Coffea canephora (Robusta coffee). The current production capacity is 3.0 million embryos per year. The pre-germination of the embryos was previously conducted by temporary immersion in liquid medium in 10-L glass bioreactors. An improved process has been developed using a 10-L disposable bioreactor consisting of a bag containing a rigid plastic box ('Box-in-Bag' bioreactor), insuring, amongst other advantages, a higher light transmittance to the biomass due to its horizontal design. For large scale cell culture, two novel flexible plastic-based disposable bioreactors have been developed from 10 to 100 L working volumes, validated with several plant species ('Wave and Undertow' and 'Slug Bubble' bioreactors). The advantages and the limits of these new types of bioreactor are discussed, based mainly on our own experience on coffee somatic embryogenesis and mass cell culture of soya and tobacco.

Genome-wide analysis of transcription factors during somatic embryogenesis in banana (Musa spp.) cv. Grand Naine.

PubMed

Shivani; Awasthi, Praveen; Sharma, Vikrant; Kaur, Navjot; Kaur, Navneet; Pandey, Pankaj; Tiwari, Siddharth

2017-01-01

Transcription factors BABY BOOM (BBM), WUSCHEL (WUS), BSD, LEAFY COTYLEDON (LEC), LEAFY COTYLEDON LIKE (LIL), VIVIPAROUS1 (VP1), CUP SHAPED COTYLEDONS (CUC), BOLITA (BOL), and AGAMOUS LIKE (AGL) play a crucial role in somatic embryogenesis. In this study, we identified eighteen genes of these nine transcription factors families from the banana genome database. All genes were analyzed for their structural features, subcellular, and chromosomal localization. Protein sequence analysis indicated the presence of characteristic conserved domains in these transcription factors. Phylogenetic analysis revealed close evolutionary relationship among most transcription factors of various monocots. The expression patterns of eighteen genes in embryogenic callus containing somatic embryos (precisely isolated by Laser Capture Microdissection), non-embryogenic callus, and cell suspension cultures of banana cultivar Grand Naine were analyzed. The application of 2, 4-dichlorophenoxyacetic acid (2, 4-D) in the callus induction medium enhanced the expression of MaBBM1, MaBBM2, MaWUS2, and MaVP1 in the embryogenic callus. It suggested 2, 4-D acts as an inducer for the expression of these genes. The higher expression of MaBBM2 and MaWUS2 in embryogenic cell suspension (ECS) as compared to non-embryogenic cells suspension (NECS), suggested that these genes may play a crucial role in banana somatic embryogenesis. MaVP1 showed higher expression in both ECS and NECS, whereas MaLEC2 expression was significantly higher in NECS. It suggests that MaLEC2 has a role in the development of non-embryogenic cells. We postulate that MaBBM2 and MaWUS2 can be served as promising molecular markers for the embryogencity in banana.

Genome-wide analysis of transcription factors during somatic embryogenesis in banana (Musa spp.) cv. Grand Naine

PubMed Central

Shivani; Awasthi, Praveen; Sharma, Vikrant; Kaur, Navjot; Kaur, Navneet; Pandey, Pankaj

2017-01-01

Transcription factors BABY BOOM (BBM), WUSCHEL (WUS), BSD, LEAFY COTYLEDON (LEC), LEAFY COTYLEDON LIKE (LIL), VIVIPAROUS1 (VP1), CUP SHAPED COTYLEDONS (CUC), BOLITA (BOL), and AGAMOUS LIKE (AGL) play a crucial role in somatic embryogenesis. In this study, we identified eighteen genes of these nine transcription factors families from the banana genome database. All genes were analyzed for their structural features, subcellular, and chromosomal localization. Protein sequence analysis indicated the presence of characteristic conserved domains in these transcription factors. Phylogenetic analysis revealed close evolutionary relationship among most transcription factors of various monocots. The expression patterns of eighteen genes in embryogenic callus containing somatic embryos (precisely isolated by Laser Capture Microdissection), non-embryogenic callus, and cell suspension cultures of banana cultivar Grand Naine were analyzed. The application of 2, 4-dichlorophenoxyacetic acid (2, 4-D) in the callus induction medium enhanced the expression of MaBBM1, MaBBM2, MaWUS2, and MaVP1 in the embryogenic callus. It suggested 2, 4-D acts as an inducer for the expression of these genes. The higher expression of MaBBM2 and MaWUS2 in embryogenic cell suspension (ECS) as compared to non-embryogenic cells suspension (NECS), suggested that these genes may play a crucial role in banana somatic embryogenesis. MaVP1 showed higher expression in both ECS and NECS, whereas MaLEC2 expression was significantly higher in NECS. It suggests that MaLEC2 has a role in the development of non-embryogenic cells. We postulate that MaBBM2 and MaWUS2 can be served as promising molecular markers for the embryogencity in banana. PMID:28797040

Identification of expressed sequences in the coffee genome potentially associated with somatic embryogenesis.

PubMed

Silva, A T; Paiva, L V; Andrade, A C; Barduche, D

2013-05-21

Brazil possesses the most modern and productive coffee growing farms in the world, but technological development is desired to cope with the increasing world demand. One way to increase Brazilian coffee growing productivity is wide scale production of clones with superior genotypes, which can be obtained with in vitro propagation technique, or from tissue culture. These procedures can generate thousands of clones. However, the methodologies for in vitro cultivation are genotype-dependent, which leads to an almost empirical development of specific protocols for each species. Therefore, molecular markers linked to the biochemical events of somatic embryogenesis would greatly facilitate the development of such protocols. In this context, sequences potentially involved in embryogenesis processes in the coffee plant were identified in silico from libraries generated by the Brazilian Coffee Genome Project. Through these in silico analyses, we identified 15 EST-contigs related to the embryogenesis process. Among these, 5 EST-contigs (3605, 9850, 13686, 17240, and 17265) could readily be associated with plant embryogenesis. Sequence analysis of EST-contig 3605, 9850, and 17265 revealed similarity to a polygalacturonase, to a cysteine-proteinase, and to an allergenine, respectively. Results also show that EST-contig 17265 sequences presented similarity to an expansin. Finally, analysis of EST-contig 17240 revealed similarity to a protein of unknown function, but it grouped in the similarity dendrogram with the WUSCHEL transcription factor. The data suggest that these EST-contigs are related to the embryogenic process and have potential as molecular markers to increase methodological efficiency in obtaining coffee plant embryogenic materials.

Transcriptome Analysis of mRNA and miRNA in Somatic Embryos of Larix leptolepis Subjected to Hydrogen Treatment.

PubMed

Liu, Yali; Han, Suying; Ding, Xiangming; Li, Xinmin; Zhang, Lifeng; Li, Wanfeng; Xu, Haiyan; Li, Zhexin; Qi, Liwang

2016-11-22

Hydrogen is a therapeutic antioxidant that has been used extensively in clinical trials. It also acts as a bioactive molecule that can alleviate abiotic stress in plants. However, the biological effects of hydrogen in somatic embryos and the underlying molecular basis remain largely unknown. In this study, the morphological and physiological influence of exogenous H₂ treatment during somatic embryogenesis was characterized in Larix leptolepis Gordon. The results showed that exposure to hydrogen increased the proportions of active pro-embryogenic cells and normal somatic embryos. We sequenced mRNA and microRNA (miRNA) libraries to identify global transcriptome changes at different time points during H₂ treatment of larch pro-embryogenic masses (PEMs). A total of 45,393 mRNAs and 315 miRNAs were obtained. Among them, 4253 genes and 96 miRNAs were differentially expressed in the hydrogen-treated libraries compared with the control. Further, a large number of the differentially expressed mRNAs and miRNAs were related to reactive oxygen species (ROS) homeostasis and cell cycle regulation. We also identified 4399 potential target genes for 285 of the miRNAs. The differential expression data and the mRNA-miRNA interaction network described here provide new insights into the molecular mechanisms that determine the performance of PEMs exposed to H₂ during somatic embryogenesis.

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.

Transcriptome Analysis of mRNA and miRNA in Somatic Embryos of Larix leptolepis Subjected to Hydrogen Treatment

PubMed Central

Liu, Yali; Han, Suying; Ding, Xiangming; Li, Xinmin; Zhang, Lifeng; Li, Wanfeng; Xu, Haiyan; Li, Zhexin; Qi, Liwang

2016-01-01

Hydrogen is a therapeutic antioxidant that has been used extensively in clinical trials. It also acts as a bioactive molecule that can alleviate abiotic stress in plants. However, the biological effects of hydrogen in somatic embryos and the underlying molecular basis remain largely unknown. In this study, the morphological and physiological influence of exogenous H2 treatment during somatic embryogenesis was characterized in Larix leptolepis Gordon. The results showed that exposure to hydrogen increased the proportions of active pro-embryogenic cells and normal somatic embryos. We sequenced mRNA and microRNA (miRNA) libraries to identify global transcriptome changes at different time points during H2 treatment of larch pro-embryogenic masses (PEMs). A total of 45,393 mRNAs and 315 miRNAs were obtained. Among them, 4253 genes and 96 miRNAs were differentially expressed in the hydrogen-treated libraries compared with the control. Further, a large number of the differentially expressed mRNAs and miRNAs were related to reactive oxygen species (ROS) homeostasis and cell cycle regulation. We also identified 4399 potential target genes for 285 of the miRNAs. The differential expression data and the mRNA-miRNA interaction network described here provide new insights into the molecular mechanisms that determine the performance of PEMs exposed to H2 during somatic embryogenesis. PMID:27879674

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.

Ectopic expression of LEAFY COTYLEDON1-LIKE gene and localized auxin accumulation mark embryogenic competence in epiphyllous plants of Helianthus annuus × H. tuberosus

PubMed Central

Chiappetta, A.; Fambrini, M.; Petrarulo, M.; Rapparini, F.; Michelotti, V.; Bruno, L.; Greco, M.; Baraldi, R.; Salvini, M.; Pugliesi, C.; Bitonti, M. B.

2009-01-01

Background and Aims The clone EMB-2 of the interspecific hybrid Helianthus annuus × H. tuberosus provides an interesting system to study molecular and physiological aspects of somatic embryogenesis. Namely, in addition to non-epiphyllous (NEP) leaves that expand normally, EMB-2 produces epiphyllous (EP) leaves bearing embryos on the adaxial surface. This clone was used to investigate if the ectopic expression of H. annuus LEAFY COTYLEDON1-LIKE (Ha-L1L) gene and auxin activity are correlated with the establishment of embryogenic competence. Methods Ha-L1L expression was evaluated by semi-quantitative RT-PCR and in situ hybridization. The endogenous level and spatial distribution of free indole-3-acetic acid (IAA) were estimated by a capillary gas chromatography–mass spectrometry–selected ion monitoring method and an immuno-cytochemical approach. Key Results Ectopic expression of Ha-L1L was detected in specific cell domains of the adaxial epidermis of EP leaves prior to the development of ectopic embryos. Ha-L1L was expressed rapidly when NEP leaves were induced to regenerate somatic embryos by in vitro culture. Differences in auxin distribution pattern rather than in absolute level were observed between EP and A-2 leaves. More precisely, a strong IAA immuno-signal was detected in single cells or in small groups of cells along the epidermis of EP leaves and accompanied the early stages of embryo development. Changes in auxin level and distribution were observed in NEP leaves induced to regenerate by in vitro culture. Exogenous auxin treatments lightly influenced Ha-L1L transcript levels in spite of an enhancement of the regeneration frequency. Conclusions In EP leaves, Ha-L1L activity marks the putative founder cells of ectopic embryos. Although the ectopic expression of Ha-L1L seems to be not directly mediated by auxin levels per se, it was demonstrated that localized Ha-L1L expression and IAA accumulation in leaf epidermis domains represent early events of somatic embryogenesis displayed by the epiphyllous EMB-2 clone. PMID:19151043

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

PubMed

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

2006-01-24

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

Somatic embryogenesis of East Kalimantan local upland rice varieties

NASA Astrophysics Data System (ADS)

Nurhasanah; Ramitha; Supriyanto, B.; Sunaryo, W.

2018-04-01

Somatic embryogenesis is the formation, growth and development of embryos from somatic cells. Somatic embryo induction is one of the in vitro plant propagation techniques that is very important for plant developmental purposes. Four local upland rice varieties of East Kalimantan, Mayas Pancing, Gedagai, Siam and Serai, were used in this study. A total of 200 explants (mature rice grains) for each varieties were inoculated on MS solid medium supplemented with 1 mg L-1 2,4 Dichlorophenoxy acetic acid (2,4-D) and 0.5 mg L-1 6-Benzylaminopurine (BAP). The results showed that response of each variety differed to embryosomatic induction, indicated by callus induction rate and callus quality, in terms of callus color and structure. The fastest callus formation was sobserved in Gedagai variety (8 days) while Mayas Pancing (13 days) was the latest one. The rate of callus induction varied from 60 to 98.5 %, and Serai variety has the highest callus induction rate. The highest friable callus structure was found in Siam variety (89.1%) and the lowest was in Gedagai (62.5%). Callus color was dominated by the yellowish-white (transparent) on all varieties tested. Most of the callus was potential as embryogenic callus characterized from the nodular and globular of friable callus structure and its yellowish-white color.

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

Protocol for in vitro somatic embryogenesis and regeneration of rice (Oryza sativa L.).

PubMed

Verma, Dipti; Joshi, Rohit; Shukla, Alok; Kumar, Pramod

2011-12-01

Development of highly efficient and reproducible plant regeneration system has tremendous potential to provide improved technology to assist in genetic transformation of indica rice cultivars for their further exploitation in selection. For the development of a highly reproducible regeneration system through somatic embryogenesis, mature embryos of highly popular rice cultivars i.e., Govind (for rainfed areas), Pusa Basmati-1 (aromatic basmati) and Jaya (for irrigated areas) were used. Optimum callus formation (%) to MS medium supplemented with 2, 4-D was obtained at 12.0 microM in Govind, 14.0 microM in Jaya and 15.0 microM in Pusa Basmati-1. All the cultivars showed good proliferation on MS medium without hormone. In Govind, highest embryogenic response was observed in MS medium supplemented with 2, 4-D (0.4 microM) + kinetin (0.4 microM), while in Pusa Basmati-1 with 2, 4-D (0.4 microM) + kinetin (2.0 microM) and in Jaya on hormone-free MS medium. Excellent embryo regeneration in Govind was observed on MS medium supplemented with low concentrations (1.1 microM) of BAP or hormone-free MS medium, while in Pusa Basmati-1 and Jaya embryogenesis was observed on MS medium supplemented with higher concentration of BAP (2.2 microM). Similarly, maximum plantlets with proliferated roots were observed in Govind on hormone-free MS medium, while in Pusa Basmati-1 and Jaya on MS medium supplemented with high concentration of NAA (4.0 microM). Developed plantlets were further successfully acclimatized and grown under pot culture up to maturity. Further the yield potential of in vitro developed plants was accessed at par to the direct seeded one under pot culture. Present, protocol standardizes somatic embryogenesis and efficient regeneration of agronomically important, high yielding and diverse indica rice cultivars which can be utilized as an efficient tool for molecular studies and genetic transformation in future.

Elongation-related functions of LEAFY COTYLEDON1 during the development of Arabidopsis thaliana.

PubMed

Junker, Astrid; Mönke, Gudrun; Rutten, Twan; Keilwagen, Jens; Seifert, Michael; Thi, Tuyet Minh Nguyen; Renou, Jean-Pierre; Balzergue, Sandrine; Viehöver, Prisca; Hähnel, Urs; Ludwig-Müller, Jutta; Altschmied, Lothar; Conrad, Udo; Weisshaar, Bernd; Bäumlein, Helmut

2012-08-01

The transcription factor LEAFY COTYLEDON1 (LEC1) controls aspects of early embryogenesis and seed maturation in Arabidopsis thaliana. To identify components of the LEC1 regulon, transgenic plants were derived in which LEC1 expression was inducible by dexamethasone treatment. The cotyledon-like leaves and swollen root tips developed by these plants contained seed-storage compounds and resemble the phenotypes produced by increased auxin levels. In agreement with this, LEC1 was found to mediate up-regulation of the auxin synthesis gene YUCCA10. Auxin accumulated primarily in the elongation zone at the root-hypocotyl junction (collet). This accumulation correlates with hypocotyl growth, which is either inhibited in LEC1-induced embryonic seedlings or stimulated in the LEC1-induced long-hypocotyl phenotype, therefore resembling etiolated seedlings. Chromatin immunoprecipitation analysis revealed a number of phytohormone- and elongation-related genes among the putative LEC1 target genes. LEC1 appears to be an integrator of various regulatory events, involving the transcription factor itself as well as light and hormone signalling, especially during somatic and early zygotic embryogenesis. Furthermore, the data suggest non-embryonic functions for LEC1 during post-germinative etiolation. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

Micropropagation of Dalbergia sissoo Roxb. through tissue culture technique.

PubMed

Sahu, Jyoti; Khan, Shagufta; Sahu, Ram Kumar; Roy, Amit

2014-04-01

Multiple shoots of Dalbergia sissoo Roxb. (Sissoo) were incited from seeds through indirect somatic embryogenesis method. Seeds were inoculated in Murashige and Skoog's medium without any growth hormone. Than cotyledonary leaves were struck and used for callus induction on MS medium amplified with 2, 4-dichlorophenoxyacetic acid (0.5 to 4 mg mL(-1)). After 3 to 4 weeks the embryogenic callus clumps was transferred to medium supplemented with cytokinin (BAP 1 to 5 mg L(-1), kinetin 1-5.0 mg L(-1)) for embryo maturation and germination. The high-frequency shoot proliferation (82%) and maximum number of shoots per explants were recorded in MS medium containing NAA (0.5)+BAP (0.5). The findings of recent investigations have shown that, it is possible to induce indirect somatic embryogenesis in Dalbergia sissoo and plant regeneration from callus cultures derived from cotyledonary leaves as explants.

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.

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

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

PubMed

Capuana, Maurizio

2016-01-01

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

Induction and cryopreservation of embryogenic cultures from nucelli and immature cotyledon cuts of mango (Mangifera indica L. var Zihua).

PubMed

Wu, Yong-Jie; Huang, Xue-Lin; Chen, Qi-Zhu; Li, Xiao-Ju; Engelmann, Florent

2007-02-01

In this paper, we described the direct somatic embryogenesis from both immature cotyledon cuts and nucelli in the same mango cultivar (Mangifera indica L. var Zihua), studied the effect of growth conditions of embryogenic cultures (EMs) on cryopreservation and compared the cryopreservation response of EMs induced from these two different explants. Histological studies demonstrated that EMs derived from nucelli could be induced directly from epidermal cells of both sides of nucelli, whereas EMs derived from cotyledon cuts were induced only from epidermal cells of the adaxial side of the cotyledons. EMs from either nucelli or cotyledon cuts could be maintained in liquid medium or on solid medium and cryopreserved using a vitrification procedure. Success of cryopreservation of EMs depended on the dehydration treatment and the defined growth conditions during culture but not on their origins. When EMs were sampled during their exponential growth phase in liquid medium and dehydrated with PVS(3) solution for 5 min, survival of the EMs induced from cotyledon cuts and nucelli reached 77.7 and 80%, respectively, after cryopreservation in liquid nitrogen for 24 h. Furthermore, when dehydrated with PVS(3) solution for 30 min, all EMs induced from cotyledon cuts and 96.7% of EMs induced from nucelli could survive after cryopreservation. Cryopreservation did not affect the plant regeneration potential of EMs through somatic embryogenesis. The protocols of somatic embryogenesis and cryopreservation of mango EMs established in this study may offer potential ways to improve mango germplasm conservation and genetic improvement.

Somatic Embryogenesis in Coffee: The Evolution of Biotechnology and the Integration of Omics Technologies Offer Great Opportunities

PubMed Central

Campos, Nádia A.; Panis, Bart; Carpentier, Sebastien C.

2017-01-01

One of the most important crops cultivated around the world is coffee. There are two main cultivated species, Coffea arabica and C. canephora. Both species are difficult to improve through conventional breeding, taking at least 20 years to produce a new cultivar. Biotechnological tools such as genetic transformation, micropropagation and somatic embryogenesis (SE) have been extensively studied in order to provide practical results for coffee improvement. While genetic transformation got many attention in the past and is booming with the CRISPR technology, micropropagation and SE are still the major bottle neck and urgently need more attention. The methodologies to induce SE and the further development of the embryos are genotype-dependent, what leads to an almost empirical development of specific protocols for each cultivar or clone. This is a serious limitation and excludes a general comprehensive understanding of the process as a whole. The aim of this review is to provide an overview of which achievements and molecular insights have been gained in (coffee) somatic embryogenesis and encourage researchers to invest further in the in vitro technology and combine it with the latest omics techniques (genomics, transcriptomics, proteomics, metabolomics, and phenomics). We conclude that the evolution of biotechnology and the integration of omics technologies offer great opportunities to (i) optimize the production process of SE and the subsequent conversion into rooted plantlets and (ii) to screen for possible somaclonal variation. However, currently the usage of the latest biotechnology did not pass the stage beyond proof of potential and needs to further improve. PMID:28871271

Gene expression and metabolite profiling of gibberellin biosynthesis during induction of somatic embryogenesis in Medicago truncatula Gaertn

PubMed Central

Igielski, Rafał

2017-01-01

Gibberellins (GAs) are involved in the regulation of numerous developmental processes in plants including zygotic embryogenesis, but their biosynthesis and role during somatic embryogenesis (SE) is mostly unknown. In this study we show that during three week- long induction phase, when cells of leaf explants from non-embryogenic genotype (M9) and embryogenic variant (M9-10a) were forming the callus, all the bioactive gibberellins from non-13-hydroxylation (GA4, GA7) and 13-hydroxylation (GA1, GA5, GA3, GA6) pathways were present, but the contents of only a few of them differed between the tested lines. The GA53 and GA19 substrates synthesized by the 13-hydroxylation pathway accumulated specifically in the M9-10a line after the first week of induction; subsequently, among the bioactive gibberellins detected, only the content of GA3 increased and appeared to be connected with acquisition of embryogenic competence. We fully annotated 20 Medicago truncatula orthologous genes coding the enzymes which catalyze all the known reactions of gibberellin biosynthesis. Our results indicate that, within all the genes tested, expression of only three: MtCPS, MtGA3ox1 and MtGA3ox2, was specific to embryogenic explants and reflected the changes observed in GA53, GA19 and GA3 contents. Moreover, by analyzing expression of MtBBM, SE marker gene, we confirmed the inhibitory effect of manipulation in GAs metabolism, applying exogenous GA3, which not only impaired the production of somatic embryos, but also significantly decreased expression of this gene. PMID:28750086

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.

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. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Effect of AgNO3 and BAP on Root as a Novel Explant in Date Palm (Phoenix dactylifera cv. Medjool) Somatic Embryogenesis.

PubMed

Roshanfekrrad, Marjan; Zarghami, Reza; Hassani, Hassan; Zakizadeh, Hedayat; Salari, Ali

2017-01-01

Somatic embryogenesis techniques are used for cloning a wide range of varieties of date palms around the world. The aim of the present study was to develop an efficient method with the lowest cost and the greatest potential to obtain in vitro plantlets of date palm cv. Medjool. Also, produce embryogenic callus and somatic embryos without using 2,4-dichlorophenoxyacetic acid (2,4-D). In this study, produced plantlets through somatic embryogenesis were used in vitro roots as explant cultured on Murashige and Skoog (MS) media containing three level of Silver Nitrate (AgNO3) (0, 3 and 6 mg L-1) plus two level of 6-benzylaminopurine (BAP) (0 and 2 mg L-1) plus 0.1 mg L-1 1-naphthylacetic acid (NAA) for callus induction. After 12 weeks of culture, callus induction and after 16 weeks, production of embryogenic callus and embryos were occurred from root explants. According to the results, medium containing 2 mg L-1 BAP and 3 mg L-1 silver nitrate+0.1 mg L-1 NAA showed the highest amount of embryogenic callus fresh weight (1.38 g). This treatment also cause the highest number and length of embryos by production of 90.04 embryogenic callus with length of 11.18 mm. On the other hand, shoots were appeared from germinated embryos and white roots began to appear within 8 weeks. Medium contains 3 mg L-1 BAP and 0.1 mg L-1 NAA with average of 12.27 cm shoot length and 15.48 cm root length was the best. Control treatment had the lowest average shoot (3.71 cm) and root (5.03 cm) length. This study showed that certain concentration of silver nitrate and BAP has stimulating effect on growth of produced embryonic callus from root segments of Medjool cultivar of date palm.

Pre-procambial cells are niches for pluripotent and totipotent stem-like cells for organogenesis and somatic embryogenesis in the peach palm: a histological study.

PubMed

de Almeida, Marcilio; de Almeida, Cristina Vieira; Mendes Graner, Erika; Ebling Brondani, Gilvano; Fiori de Abreu-Tarazi, Monita

2012-08-01

The direct induction of adventitious buds and somatic embryos from explants is a morphogenetic process that is under the influence of exogenous plant growth regulators and its interactions with endogenous phytohormones. We performed an in vitro histological analysis in peach palm (Bactris gasipaes Kunth) shoot apexes and determined that the positioning of competent cells and their interaction with neighboring cells, under the influence of combinations of exogenously applied growth regulators (NAA/BAP and NAA/TDZ), allows the pre-procambial cells (PPCs) to act in different morphogenic pathways to establish niche competent cells. It is likely that there has been a habituation phenomenon during the regeneration and development of the microplants. This includes promoting the tillering of primary or secondary buds due to culturing in the absence of NAA/BAP or NAA/TDZ after a period in the presence of these growth regulators. Histological analyses determined that the adventitious roots were derived from the dedifferentiation of the parenchymal cells located in the basal region of the adventitious buds, with the establishment of rooting pole, due to an auxin gradient. Furthermore, histological and histochemical analyses allowed us to characterize how the PPCs provide niches for multipotent, pluripotent and totipotent stem-like cells for vascular differentiation, organogenesis and somatic embryogenesis in the peach palm. The histological and histochemical analyses also allowed us to detect the unicellular or multicellular origin of somatic embryogenesis. Therefore, our results indicate that the use of growth regulators in microplants can lead to habituation and to different morphogenic pathways leading to potential niche establishment, depending on the positioning of the competent cells and their interaction with neighboring cells. Our results indicate that the use of growth regulators in microplants can lead to habituation and to different morphogenic pathways leading to potential niche establishment, depending on the positioning of the competent cells and their interaction with neighboring cells.

Heritability of regeneration in tissue cultures of sweet potato (Ipomoea batatas L.).

PubMed

Templeton-Somers, K M; Collins, W W

1986-03-01

A population of open-pollinated progeny from 12 parents, and the 12 parents, was surveyed for in vitro growth and regeneration characteristics. Four different tissue culture procedures involving different media and the use of different explants to initiate the cultures were used. Petiole explants from young leaves were used as explants for initiation of callus cultures. These were evaluated for callus growth rate, friability, and callus color and texture, before transferring to each of three different regeneration media for evaluation of morphogenetic potential. Small shoot tips also were used to initiate callus cultures, which were evaluated for the same growth characteristics and transferred to growth-regulator free regeneration media. Regeneration occurred through root or shoot regeneration or through embryogenesis. Tissue culture treatment effects, as well as genotypic effects, were highly significant in determining: the types of callus produced, callus growth rates, color and texture on the two types of media used for the second and third subcultures. The family x treatment interaction was generally not statistically significant, affecting only callus color. Estimates of narrow sense heritability for callus growth rate in both the second and third subcultures were high enough (0.35 and 0.63, respectively) for the evaluation of parental lines for selection procedures. These characteristics were also the only early culture callus traits that were consistently correlated with later morphogenesis of the cultures. They were negatively correlated with root or shoot regeneration. The occurence of somatic embryogenesis was not correlated with early callus growth characteristics. Genetic and treatment effects were highly significant in the evaluation of morphogenetic potential, through root or shoot regeneration, or through embryogenesis. Regeneration of all types was of low frequency for all procedures, expressed in ≦ 11% of the cultures of the total population.

Biolistic transformation of cotton embryogenic cell suspension cultures

USDA-ARS?s Scientific Manuscript database

Genetic transformation of cotton is highly dependent on the ability to regenerate fertile plants from transgenic cells through somatic embryogenesis. Induction of embryogenic cell cultures is genotype-dependant. However, once embryogenic cell cultures are available, they can be effectively used fo...

Control of root meristem establishment in conifers.

PubMed

Brunoni, Federica; Ljung, Karin; Bellini, Catherine

2018-06-19

The evolution of terrestrial plant life was made possible by the establishment of a root system, which enabled plants to migrate from aquatic to terrestrial habitats. During evolution, root organization has gradually progressed from a very simple to a highly hierarchical architecture. Roots are initiated during embryogenesis and branch afterwards through lateral root formation. Additionally, adventitious roots can be formed post-embryonically from aerial organs. Induction of adventitious roots forms the basis of the vegetative propagation via cuttings in horticulture, agriculture and forestry. This method, together with somatic embryogenesis, is routinely used to clonally multiply conifers. In addition to being utilized as propagation techniques, adventitious rooting and somatic embryogenesis have emerged as versatile models to study cellular and molecular mechanisms of embryo formation and organogenesis of coniferous species. Both formation of the embryonic root and the adventitious root primordia require the establishment of auxin gradients within cells that coordinate the developmental response. These processes also share key elements of the genetic regulatory networks that, for example, are triggering cell fate. This minireview gives an overview of the molecular control mechanisms associated with root development in conifers, from initiation in the embryo to post-embryonic formation in cuttings. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Ectopic expression of Triticum aestivum SERK genes (TaSERKs) control plant growth and development in Arabidopsis.

PubMed

Singh, Akanksha; Khurana, Paramjit

2017-09-28

Somatic embryogenesis receptor kinases (SERKs) belong to a small gene family of receptor-like kinases involved in signal transduction. A total of 54 genes were shortlisted from the wheat genome survey sequence of which 5 were classified as SERKs and 49 were identified as SERK-like (SERLs). Tissue- specific expression of TaSERKs at major developmental stages of wheat corroborates their indispensable role during somatic and zygotic embryogenesis. TaSERK transcripts show inherent differences in their hormonal sensitivities, i.e. TaSERK2 and TaSERK3 elicits auxin- specific responses while TaSERK1, 4 and 5 were more specific towards BR-mediated regulation. The ectopic expression of TaSERK1, 2, 3, 4 and 5 in Arabidopsis led to enhanced plant height, larger silique size and increased seed yield. Zygotic embryogenesis specific genes showed a differential pattern in TaSERK Arabidopsis transgenics specifically in the silique tissues. Elongated hypocotyls and enhanced root growth were observed in the overexpression transgenic lines of all five TaSERKs. The inhibitory action of auxin and brassinosteroid in all the TaSERK transgenic lines indicates their role in regulating root development. The results obtained imply redundant functions of TaSERKs in maintaining plant growth and development.

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

Maize embryogenesis.

PubMed

Fontanet, Pilar; Vicient, Carlos M

2008-01-01

Plant embryo development is a complex process that includes several coordinated events. Maize mature embryos consist of a well-differentiated embryonic axis surrounded by a single massive cotyledon called scutellum. Mature embryo axis also includes lateral roots and several developed leaves. In contrast to Arabidopsis, in which the orientation of cell divisions are perfectly established, only the first planes of cell division are predictable in maize embryos. These distinctive characteristics joined to the availability of a large collection of embryo mutants, well-developed molecular biology and tissue culture tools, an established genetics and its economical importance make maize a good model plant for grass embryogenesis. Here, we describe basic concepts and techniques necessary for studying maize embryo development: how to grow maize in greenhouses and basic techniques for in vitro embryo culture, somatic embryogenesis and in situ hybridization.

Biotechnology of trees: Chestnut

Treesearch

C.D. Nelson; W.A. Powell; S.A. Merkle; J.E. Carlson; F.V. Hebard; N Islam-Faridi; M.E. Staton; L. Georgi

2014-01-01

Biotechnology has been practiced on chestnuts (Castanea spp.) for many decades, including vegetative propagation, controlled crossing followed by testing and selection, genetic and cytogenetic mapping, genetic modifi cation, and gene and genome sequencing. Vegetative propagation methods have ranged from grafting and rooting to somatic embryogenesis, often in...

Somatic embryogenesis from immature fruit of Juglans cinerea

Treesearch

Paula M. Pijut

1999-01-01

Butternut (Juglans cinerea L.) (Fig. 1a), also known as white walnut or oilnut, is a hardwood species in the family Juglandaceae, section Trachycaryon (Manning, 1978), or more appropriately, section Cardiocaryon (Fjellstrom & Parfitt, 1994). This hardwood species is valued for its wood and edible...

Micropropagation, genetic engineering, and molecular biology of Populus

Treesearch

N. B. Klopfenstein; Y. W. Chun; M. -S. Kim; M. A. Ahuja; M. C. Dillon; R. C. Carman; L. G. Eskew

1997-01-01

Thirty-four Populus biotechnology chapters, written by 85 authors, are comprised in 5 sections: 1) in vitro culture (micropropagation, somatic embryogenesis, protoplasts, somaclonal variation, and germplasm preservation); 2) transformation and foreign gene expression; 3) molecular biology (molecular/genetic characterization); 4) biotic and abiotic resistance (disease,...

In Vitro Mass Propagation of Cymbopogon citratus Stapf., a Medicinal Gramineae.

PubMed

Quiala, Elisa; Barbón, Raúl; Capote, Alina; Pérez, Naivy; Jiménez, Elio

2016-01-01

Cymbopogon citratus (D.C.) Stapf. is a medicinal plant source of lemon grass oils with multiple uses in the pharmaceutical and food industry. Conventional propagation in semisolid culture medium has become a fast tool for mass propagation of lemon grass, but the production cost must be lower. A solution could be the application of in vitro propagation methods based on liquid culture advantages and automation. This chapter provides two efficient protocols for in vitro propagation via organogenesis and somatic embryogenesis of this medicinal plant. Firstly, we report the production of shoots using a temporary immersion system (TIS). Secondly, a protocol for somatic embryogenesis using semisolid culture for callus formation and multiplication, and liquid culture in a rotatory shaker and conventional bioreactors for the maintenance of embryogenic culture, is described. Well-developed plants can be achieved from both protocols. Here we provide a fast and efficient technology for mass propagation of this medicinal plant taking the advantage of liquid culture and automation.

Carbon source dependent somatic embryogenesis and plant regeneration in cotton, Gossypium hirsutum L. cv. SVPR2 through suspension cultures.

PubMed

Ganesan, M; Jayabalan, N

2005-10-01

Highly reproducible and simple protocol for cotton somatic embryogenesis is described here by using different concentrations of maltose, glucose, sucrose and fructose. Maltose (30 g/l) is the best carbon source for embryogenic callus induction and glucose (30 g/l) was suitable for induction, maturation of embryoids and plant regeneration. Creamy white embryogenic calli of hypocotyl explants were formed on medium containing MS basal salts, myo-inositol (100 mg/l), thiamine HCI (0.3 mg/l), picloram (0.3 mg/l), Kin (0.1 mg/l) and maltose (30 g/l). During embryo induction and maturation, accelerated growth was observed in liquid medium containing NH3NO4 (1 g/l), picloram (2.0 mg/l), 2 ip (0.2 mg/l), Kin (0.1 mg/l) and glucose (30 g/l). Before embryoid induction, large clumps of embryogenic tissue were formed. These tissues only produced viable embryoids. Completely matured somatic embryos were germinated successfully on the medium fortified with MS salts, myo-inositol (50 mg/l), thiamine HCl (0.2 mg/l), GA3 (0.2 mg/l), BA (1.0 mg/l) and glucose (30 g/l). Compared with earlier reports, 65% of somatic embryo germination was observed. The abnormal embryo formation was highly reduced by using glucose (30 g/l) compared to other carbon sources. The regenerated plantlets were fertile but smaller in height than the seed derived control plants.

Embling Production in Althaea officinalis L., Through Somatic Embryogenesis and Their Appraisal via Histological and Scanning Electron Microscopical Studies.

PubMed

Naz, Ruphi; Anis, Mohammad; Alatar, Abdulrahman A

2017-07-01

In vitro propagation of a medicinally important plant, Althaea officinalis, has been achieved through somatic embryogenesis. Somatic embryos (globular to torpedo-shaped embryos) were induced on Murashige and Skoog's (MS) medium augmented with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D, 5.0, 10.0, 15.0, 20.0, and 25.0) alone or combined with N6-benzylaminopurine (BA, 0.1, 0.5, 1.0, 1.5, and 2.0 μM). These were directly formed from the cut ends and subsequently spread on the whole surface of internodal explants. For embryo maturation, torpedo embryos were transferred on a medium containing different levels of BA (0.1, 0.5, or 1.0 μM) and abscisic acid (ABA) (0.5, 1.0, or 1.5 μM) or α-naphthalene acetic acid (NAA) (0.1, 0.5 or 1.0 μM). Among the different concentrations tested, 0.5 μM BA along with 1.0 μM ABA was found most effective, on which a highest yield (58.0%) with an optimum number (35.0) of mature embryos (cotyledonary stage) was observed after 2 weeks of transfer. Germination of cotyledonary embryos into plantlets with 68% were observed on ½ MS medium. Histological and scanning electron microscopical (SEM) studies proved that the regenerated structures were somatic embryos and not shoot primordia. Plants grew vigorously when transferred to a greenhouse.

Biotechnological advances in mango (Mangifera indica L.) and their future implication in crop improvement: a review.

PubMed

Krishna, Hare; Singh, S K

2007-01-01

Biotechnology can complement conventional breeding and expedite the mango improvement programmes. Studies involving in vitro culture and selection, micropropagation, embryo rescue, genetic transformation, marker-assisted characterization and DNA fingerprinting, etc. are underway at different centers worldwide. In vitro culture and somatic embryogenesis of several different genotypes have been achieved. The nucellus excised from immature fruitlets is the appropriate explant for induction of embryogenic cultures. High frequency somatic embryogenesis has been achieved in some genotypes; however, some abnormalities can occur during somatic embryo germination. Embryo rescue from young and dropped fruitlets can improve the hybridization success in a limited flowering season. Protocols for protoplast culture and regeneration have also been developed. In vitro selections for antibiotic tolerance and fungal toxin resistance have been very promising for germplasm screening. Genetic transformation using Agrobacterium tumefaciens has been reported. Genes that are involved with fruit ripening have been cloned and there have been attempts to deliver these genes into plants. DNA fingerprinting and studies on genetic diversity of mango cultivars and Mangifera species are also being conducted at several research stations. The purpose of this review is to focus upon contemporary information on biotechnological advances made in mango. It also describes some ways of overcoming the problems encountered during in vitro propagation of mango.

Role of polyamines in somatic embryogenesis

Treesearch

S.C. Minocha; R. Minocha

1995-01-01

The aliphatic amines putrescine, spermidine, and spermine are present in all living organisms. Since the demonstration of "an essential nutritional function" for putrescine in the bacterium Hemophilus parainfluenzae (Herbst and Snell 1948), polyamines have attracted a great deal of attention from workers in diverse fields of the life...

An Efficient In Vitro Regeneration System for Ornamental Ginger (Hedychium spp.)

USDA-ARS?s Scientific Manuscript database

An improved and efficient regeneration protocol was established for Hedychium via somatic embryogenesis. The plant material used consisted of 11 species and 9 cultivars of Hedychium. The explants consisted of young leaves taken from lateral or terminal shoots of mature greenhouse grown plants. These...

EMBRYONIC DEVELOPMENT AND A QUANTITATIVE MODEL OF PROGRAMMED DNA ELIMINATION IN MESOCYCLOPS EDAX (S. A. FORBES, 1891) (COPEPODA: CYCLOPOIDA)

PubMed Central

Clower, Michelle K.; Holub, Ashton S.; Smith, Rebecca T.; Wyngaard, Grace A.

2016-01-01

The highly programmed fragmentation of chromosomes and elimination of large amounts of nuclear DNA from the presomatic cell lineages (i.e., chromatin diminution), occurs in the embryos of the freshwater zooplankton Mesocyclops edax (S. A. Forbes, 1891) (Crustacea: Copepoda). The somatic genome is reorganized and reduced to a size five times smaller even though the germline genome remains intact. We present the first comprehensive, quantitative model of DNA content throughout embryogenesis in a copepod that possesses embryonic DNA elimination. We used densitometric image analysis to measure the DNA content of polar bodies, germline and somatic nuclei, and excised DNA “droplets.” We report: 1) variable DNA contents of polar bodies, some of which do not contain the amount corresponding to the haploid germline genome size; 2) presence of pronuclei in newly laid embryo sacs; 3) gonomeric chromosomes in the second to fourth cleavage divisions and in the primordial germ cell and primordial endoderm cell during the fifth cleavage division; 4) timing of early embryonic cell stages, elimination of DNA, and divisions of the primordial germ cell and primordial endoderm cell at 22°C; and 5) persistence of a portion of the excised DNA “droplets” throughout embryogenesis. DNA elimination is a trait that spans multiple embryonic stages and a knowledge of the timing and variability of the associated cytological events with DNA elimination will promote the study of the molecular mechanisms involved in this trait. We propose the “genome yolk hypothesis” as a functional explanation for the persistence of the eliminated DNA that might serve as a resource during postdiminution cleavage divisions. PMID:27857452

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 onsetmore » 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.« less

Identification of novel genes potentially involved in somatic embryogenesis in chicory (Cichorium intybus L.)

PubMed Central

2010-01-01

Background In our laboratory we use cultured chicory (Cichorium intybus) explants as a model to investigate cell reactivation and somatic embryogenesis and have produced 2 chicory genotypes (K59, C15) sharing a similar genetic background. K59 is a responsive genotype (embryogenic) capable of undergoing complete cell reactivation i.e. cell de- and re-differentiation leading to somatic embryogenesis (SE), whereas C15 is a non-responsive genotype (non-embryogenic) and is unable to undergo SE. Previous studies [1] showed that the use of the β-D-glucosyl Yariv reagent (β-GlcY) that specifically binds arabinogalactan-proteins (AGPs) blocked somatic embryo production in chicory root explants. This observation indicates that β-GlcY is a useful tool for investigating somatic embryogenesis (SE) in chicory. In addition, a putative AGP (DT212818) encoding gene was previously found to be significantly up-regulated in the embryogenic K59 chicory genotype as compared to the non-embryogenic C15 genotype suggesting that this AGP could be involved in chicory re-differentiation [2]. In order to improve our understanding of the molecular and cellular regulation underlying SE in chicory, we undertook a detailed cytological study of cell reactivation events in K59 and C15 genotypes, and used microarray profiling to compare gene expression in these 2 genotypes. In addition we also used β-GlcY to block SE in order to identify genes potentially involved in this process. Results Microscopy confirmed that only the K59, but not the C15 genotype underwent complete cell reactivation leading to SE formation. β-GlcY-treatment of explants blocked in vitro SE induction, but not cell reactivation, and induced cell wall modifications. Microarray analyses revealed that 78 genes were differentially expressed between induced K59 and C15 genotypes. The expression profiles of 19 genes were modified by β-GlcY-treatment. Eight genes were both differentially expressed between K59 and C15 genotypes during SE induction and transcriptionally affected by β-GlcY-treatment: AGP (DT212818), 26 S proteasome AAA ATPase subunit 6 (RPT6), remorin (REM), metallothionein-1 (MT1), two non-specific lipid transfer proteins genes (SDI-9 and DEA1), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), and snakin 2 (SN2). These results suggest that the 8 genes, including the previously-identified AGP gene (DT212818), could be involved in cell fate determination events leading to SE commitment in chicory. Conclusion The use of two different chicory genotypes differing in their responsiveness to SE induction, together with β-GlcY-treatment represented an efficient tool to discriminate cell reactivation from the SE morphogenetic pathway. Such an approach, together with microarray analyses, permitted us to identify several putative key genes related to the SE morphogenetic pathway in chicory. PMID:20565992

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

PubMed Central

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

2007-01-01

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

Mammalian pre-implantation chromosomal instability: species comparison, evolutionary considerations, and pathological correlations.

PubMed

Carbone, Lucia; Chavez, Shawn L

2015-01-01

Pre-implantation embryo development in mammals begins at fertilization with the migration and fusion of the maternal and paternal pro-nuclei, followed by the degradation of inherited factors involved in germ cell specification and the activation of embryonic genes required for subsequent cell divisions, compaction, and blastulation. The majority of studies on early embryogenesis have been conducted in the mouse or non-mammalian species, often requiring extrapolation of the findings to human development. Given both conserved similarities and species-specific differences, however, even comparison between closely related mammalian species may be challenging as certain aspects, including susceptibility to chromosomal aberrations, varies considerably across mammals. Moreover, most human embryo studies are limited to patient samples obtained from in vitro fertilization (IVF) clinics and donated for research, which are generally of poorer quality and produced with germ cells that may be sub-optimal. Recent technical advances in genetic, epigenetic, chromosomal, and time-lapse imaging analyses of high quality whole human embryos have greatly improved our understanding of early human embryogenesis, particularly at the single embryo and cell level. This review summarizes the major characteristics of mammalian pre-implantation development from a chromosomal perspective, in addition to discussing the technological achievements that have recently been developed to obtain this data. We also discuss potential translation to clinical applications in reproductive medicine and conclude by examining the broader implications of these findings for the evolution of mammalian species and cancer pathology in somatic cells.

Proteome reference maps of Medicago truncatula embryogenic cell cultures generated from single protoplasts.

PubMed

Imin, Nijat; De Jong, Femke; Mathesius, Ulrike; van Noorden, Giel; Saeed, Nasir A; Wang, Xin-Ding; Rose, Ray J; Rolfe, Barry G

2004-07-01

Using a combination of two-dimensional gel electrophoresis (2-DE) protein mapping and mass spectrometry (MS) analysis, we have established proteome reference maps of Medicago truncatula embryogenic tissue culture cells. The cultures were generated from single protoplasts, which provided a relatively homogeneous cell population. We used these to analyze protein expression at the globular stages of somatic embryogenesis, which is the earliest morphogenetic embryonic stage. Over 3000 proteins could reproducibly be resolved over a pI range of 4-11. Three hundred and twelve protein spots were extracted from colloidal Coomassie Blue-stained 2-DE gels and analyzed by matrix-assisted laser desorption/ionization-time of flight MS analysis and tandem MS sequencing. This enabled the identification of 169 protein spots representing 128 unique gene products using a publicly available expressed sequence tag database and the MASCOT search engine. These reference maps will be valuable for the investigation of the molecular events which occur during somatic embryogenesis in M. truncatula. The proteome reference maps and supplementary materials will be available and updated for public access at http://semele.anu.edu.au/.

The Use of Proteomic Tools to Address Challenges Faced in Clonal Propagation of Tropical Crops through Somatic Embryogenesis.

PubMed

Chin, Chiew Foan; Tan, Hooi Sin

2018-05-04

In many tropical countries with agriculture as the mainstay of the economy, tropical crops are commonly cultivated at the plantation scale. The successful establishment of crop plantations depends on the availability of a large quantity of elite seedling plants. Many plantation companies establish plant tissue culture laboratories to supply planting materials for their plantations and one of the most common applications of plant tissue culture is the mass propagation of true-to-type elite seedlings. However, problems encountered in tissue culture technology prevent its applications being widely adopted. Proteomics can be a powerful tool for use in the analysis of cultures, and to understand the biological processes that takes place at the cellular and molecular levels in order to address these problems. This mini review presents the tissue culture technologies commonly used in the propagation of tropical crops. It provides an outline of some the genes and proteins isolated that are associated with somatic embryogenesis and the use of proteomic technology in analysing tissue culture samples and processes in tropical crops.

DNA methylome of the 20-gigabase Norway spruce genome

PubMed Central

Ausin, Israel; Feng, Suhua; Yu, Chaowei; Liu, Wanlu; Kuo, Hsuan Yu; Jacobsen, Elise L.; Zhai, Jixian; Gallego-Bartolome, Javier; Wang, Lin; Egertsdotter, Ulrika; Street, Nathaniel R.; Jacobsen, Steven E.; Wang, Haifeng

2016-01-01

DNA methylation plays important roles in many biological processes, such as silencing of transposable elements, imprinting, and regulating gene expression. Many studies of DNA methylation have shown its essential roles in angiosperms (flowering plants). However, few studies have examined the roles and patterns of DNA methylation in gymnosperms. Here, we present genome-wide high coverage single-base resolution methylation maps of Norway spruce (Picea abies) from both needles and somatic embryogenesis culture cells via whole genome bisulfite sequencing. On average, DNA methylation levels of CG and CHG of Norway spruce were higher than most other plants studied. CHH methylation was found at a relatively low level; however, at least one copy of most of the RNA-directed DNA methylation pathway genes was found in Norway spruce, and CHH methylation was correlated with levels of siRNAs. In comparison with needles, somatic embryogenesis culture cells that are used for clonally propagating spruce trees showed lower levels of CG and CHG methylation but higher level of CHH methylation, suggesting that like in other species, these culture cells show abnormal methylation patterns. PMID:27911846

Identification of differentially accumulated proteins associated with embryogenic and non-embryogenic calli in saffron (Crocus sativus L.)

PubMed Central

2012-01-01

Background Somatic embryogenesis (SE) is a complex biological process that occurs under inductive conditions and causes fully differentiated cells to be reprogrammed to an embryo like state. In order to get a better insight about molecular basis of the SE in Crocus sativus L. and to characterize differentially accumulated proteins during the process, a proteomic study based on two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry has been carried out. Results We have compared proteome profiles of non-embryogenic and embryogenic calli with native corm explants. Total soluble proteins were phenol-extracted and loaded on 18 cm IPG strips for the first dimension and 11.5% sodium dodecyl sulfate-polyacrylamide gels for the second dimension. Fifty spots with more than 1.5-fold change in abundance were subjected to mass spectrometry analysis for further characterization. Among them 36 proteins could be identified, which are classified into defense and stress response, protein synthesis and processing, carbohydrate and energy metabolism, secondary metabolism, and nitrogen metabolism. Conclusion Our results showed that diverse cellular and molecular processes were affected during somatic to embryogenic transition. Differential proteomic analysis suggests a key role for ascorbate metabolism during early stage of SE, and points to the possible role of ascorbate-glutathione cycle in establishing somatic embryos. PMID:22243837

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.

Somatic embryogenesis from seeds in a broad range of Vitis vinifera L. varieties: rescue of true-to-type virus-free plants.

PubMed

San Pedro, Tània; Gammoudi, Najet; Peiró, Rosa; Olmos, Antonio; Gisbert, Carmina

2017-11-29

Somatic embryogenesis is the preferred method for cell to plant regeneration in Vitis vinifera L. However, low frequencies of plant embryo conversion are commonly found. In a previous work we obtained from cut-seeds of a grapevine infected with the Grapevine leafroll associated viruses 1 and 3 (GLRaV-1 and GLRaV-3), high rates of direct regeneration, embryo plant conversion and sanitation. The aim of this study is to evaluate the usefulness of this procedure for regeneration of other grapevine varieties which include some infected with one to three common grapevine viruses (GLRaV-3, Grapevine fanleaf virus (GFLV) and Grapevine fleck virus (GFkV)). As grapevine is highly heterozygous, it was necessary to select from among the virus-free plants those that regenerated from mother tissues around the embryo, (true-to-type). Somatic embryogenesis and plant regeneration were achieved in a first experiment, using cut-seeds from the 14 grapevine varieties Airén, Cabernet Franc, Cabernet Sauvignon, Mencía, Merlot, Monastrell, Petit Verdot, Pinot Blanc (infected by GFLV and GFkV), Pinot Gris, Pinot Meunier, Pinot Noir, Syrah, Tempranillo (infected by GFLV), and Verdil. All regenerated plants were confirmed to be free of GFkV whereas at least 68% sanitation was obtained for GFLV. The SSR profiles of the virus-free plants showed, in both varieties, around 10% regeneration from mother tissue (the same genetic make-up as the mother plant). In a second experiment, this procedure was used to sanitize the varieties Cabernet Franc, Godello, Merlot and Valencí Blanc infected by GLRaV-3, GFkV and/or GFLV. Cut-seeds can be used as explants for embryogenesis induction and plant conversion in a broad range of grapevine varieties. The high regeneration rates obtained with this procedure facilitate the posterior selection of true-to-type virus-free plants. A sanitation rate of 100% was obtained for GFkV as this virus is not seed-transmitted. However, the presence of GLRaV-3 and GFLV in some of the regenerated plants showed that both viruses are seed-transmitted. The regeneration of true-to-type virus-free plants from all infected varieties indicates that this methodology may represent an alternative procedure for virus cleaning in grapevine.

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

PubMed Central

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

2013-01-01

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

Transcriptomic analysis highlights epigenetic and transcriptional regulation during zygotic embryo development of Pinus pinaster.

PubMed

de Vega-Bartol, José J; Simões, Marta; Lorenz, W Walter; Rodrigues, Andreia S; Alba, Rob; Dean, Jeffrey F D; Miguel, Célia M

2013-08-30

It is during embryogenesis that the plant body plan is established and the meristems responsible for all post-embryonic growth are specified. The molecular mechanisms governing conifer embryogenesis are still largely unknown. Their elucidation may contribute valuable information to clarify if the distinct features of embryo development in angiosperms and gymnosperms result from differential gene regulation. To address this issue, we have performed the first transcriptomic analysis of zygotic embryo development in a conifer species (Pinus pinaster) focusing our study in particular on regulatory genes playing important roles during plant embryo development, namely epigenetic regulators and transcription factors. Microarray analysis of P. pinaster zygotic embryogenesis was performed at five periods of embryo development from early developing to mature embryos. Our results show that most changes in transcript levels occurred in the first and the last embryo stage-to-stage transitions, namely early to pre-cotyledonary embryo and cotyledonary to mature embryo. An analysis of functional categories for genes that were differentially expressed through embryogenesis highlighted several epigenetic regulation mechanisms. While putative orthologs of transcripts associated with mechanisms that target transposable elements and repetitive sequences were strongly expressed in early embryogenesis, PRC2-mediated repression of genes seemed more relevant during late embryogenesis. On the other hand, functions related to sRNA pathways appeared differentially regulated across all stages of embryo development with a prevalence of miRNA functions in mid to late embryogenesis. Identification of putative transcription factor genes differentially regulated between consecutive embryo stages was strongly suggestive of the relevance of auxin responses and regulation of auxin carriers during early embryogenesis. Such responses could be involved in establishing embryo patterning. Later in development, transcripts with homology to genes acting on modulation of auxin flow and determination of adaxial-abaxial polarity were up-regulated, as were putative orthologs of genes required for meristem formation and function as well as establishment of organ boundaries. Comparative analysis with A. thaliana embryogenesis also highlighted genes involved in auxin-mediated responses, as well as epigenetic regulation, indicating highly correlated transcript profiles between the two species. This is the first report of a time-course transcriptomic analysis of zygotic embryogenesis in a conifer. Taken together our results show that epigenetic regulation and transcriptional control related to auxin transport and response are critical during early to mid stages of pine embryogenesis and that important events during embryogenesis seem to be coordinated by putative orthologs of major developmental regulators in angiosperms.

Transcriptomic analysis highlights epigenetic and transcriptional regulation during zygotic embryo development of Pinus pinaster

PubMed Central

2013-01-01

Background It is during embryogenesis that the plant body plan is established and the meristems responsible for all post-embryonic growth are specified. The molecular mechanisms governing conifer embryogenesis are still largely unknown. Their elucidation may contribute valuable information to clarify if the distinct features of embryo development in angiosperms and gymnosperms result from differential gene regulation. To address this issue, we have performed the first transcriptomic analysis of zygotic embryo development in a conifer species (Pinus pinaster) focusing our study in particular on regulatory genes playing important roles during plant embryo development, namely epigenetic regulators and transcription factors. Results Microarray analysis of P. pinaster zygotic embryogenesis was performed at five periods of embryo development from early developing to mature embryos. Our results show that most changes in transcript levels occurred in the first and the last embryo stage-to-stage transitions, namely early to pre-cotyledonary embryo and cotyledonary to mature embryo. An analysis of functional categories for genes that were differentially expressed through embryogenesis highlighted several epigenetic regulation mechanisms. While putative orthologs of transcripts associated with mechanisms that target transposable elements and repetitive sequences were strongly expressed in early embryogenesis, PRC2-mediated repression of genes seemed more relevant during late embryogenesis. On the other hand, functions related to sRNA pathways appeared differentially regulated across all stages of embryo development with a prevalence of miRNA functions in mid to late embryogenesis. Identification of putative transcription factor genes differentially regulated between consecutive embryo stages was strongly suggestive of the relevance of auxin responses and regulation of auxin carriers during early embryogenesis. Such responses could be involved in establishing embryo patterning. Later in development, transcripts with homology to genes acting on modulation of auxin flow and determination of adaxial-abaxial polarity were up-regulated, as were putative orthologs of genes required for meristem formation and function as well as establishment of organ boundaries. Comparative analysis with A. thaliana embryogenesis also highlighted genes involved in auxin-mediated responses, as well as epigenetic regulation, indicating highly correlated transcript profiles between the two species. Conclusions This is the first report of a time-course transcriptomic analysis of zygotic embryogenesis in a conifer. Taken together our results show that epigenetic regulation and transcriptional control related to auxin transport and response are critical during early to mid stages of pine embryogenesis and that important events during embryogenesis seem to be coordinated by putative orthologs of major developmental regulators in angiosperms. PMID:23987738

An Organismal CNV Mutator Phenotype Restricted to Early Human Development.

PubMed

Liu, Pengfei; Yuan, Bo; Carvalho, Claudia M B; Wuster, Arthur; Walter, Klaudia; Zhang, Ling; Gambin, Tomasz; Chong, Zechen; Campbell, Ian M; Coban Akdemir, Zeynep; Gelowani, Violet; Writzl, Karin; Bacino, Carlos A; Lindsay, Sarah J; Withers, Marjorie; Gonzaga-Jauregui, Claudia; Wiszniewska, Joanna; Scull, Jennifer; Stankiewicz, Paweł; Jhangiani, Shalini N; Muzny, Donna M; Zhang, Feng; Chen, Ken; Gibbs, Richard A; Rautenstrauss, Bernd; Cheung, Sau Wai; Smith, Janice; Breman, Amy; Shaw, Chad A; Patel, Ankita; Hurles, Matthew E; Lupski, James R

2017-02-23

De novo copy number variants (dnCNVs) arising at multiple loci in a personal genome have usually been considered to reflect cancer somatic genomic instabilities. We describe a multiple dnCNV (MdnCNV) phenomenon in which individuals with genomic disorders carry five to ten constitutional dnCNVs. These CNVs originate from independent formation incidences, are predominantly tandem duplications or complex gains, exhibit breakpoint junction features reminiscent of replicative repair, and show increased de novo point mutations flanking the rearrangement junctions. The active CNV mutation shower appears to be restricted to a transient perizygotic period. We propose that a defect in the CNV formation process is responsible for the "CNV-mutator state," and this state is dampened after early embryogenesis. The constitutional MdnCNV phenomenon resembles chromosomal instability in various cancers. Investigations of this phenomenon may provide unique access to understanding genomic disorders, structural variant mutagenesis, human evolution, and cancer biology. Copyright © 2017 Elsevier Inc. All rights reserved.

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. Copyright © 2014 Elsevier Inc. All rights reserved.

Somatic embryogenesis tissue culture for applying varietal forestry to conifer species

Treesearch

Steven C. Grossnickle; John Pait

2008-01-01

The use of tree improvement practices to enhance the genetic characteristics of planted seedlings is a forestry practice that consistently shows a high return on investment by increasing yields obtained from planted forests. The use of improved seeds is an effective way of bringing genetic improvement to forest regeneration programs. Seed orchards are currently used to...

Historical aspects of somatic embryogenesis in woody plants

Treesearch

Subhash C. Minocha; Rakesh Minocha

1995-01-01

During the next few decades, the world demand for wood products is expected to rise sharply. To meet this growing demand, there will be an increasing need for mass production of improved-quality planting stock of many tree species. The conventional methods of tree improvement and selection offer only limited possibility of meeting the growing demands. Therefore, new...

Somatic embryogenesis and cryostorage for conservation and restoration of threatened forest trees

Treesearch

S.A. Merkle; A.R. Tull; H.J. Gladfelter; P.M. Montello; J.E. Mitchell; C. Ahn; R.D. McNeill

2017-01-01

Threats to North American forest trees from exotic pests and pathogens or habitat loss, make it imperative that every available tool be employed for conservation and restoration of these at risk species. One such tool, in vitro propagation, could greatly enhance conservation of forest tree genetic material and selection and breeding of resistant or...

Inducing Somatic Pkd1 Mutations in Vivo in a Mouse Model of Autosomal-Dominant Polycystic Kidney Disease

DTIC Science & Technology

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the worlds most common life threatening genetic diseases. Over 95 percent of diagnosed...several genetic models to induce mutations: two during embryogenesis (with Six2-cre and CVM-cre) and one in the adult (Villin-cre). One of the embryonic

Search for methylation-sensitive amplification polymorphisms associated with the mantled variant phenotype in oil palm (Elaeis guineensis Jacq).

PubMed

Jaligot, E; Beulé, T; Baurens, F-C; Billotte, N; Rival, A

2004-02-01

The methylation-sensitive amplification polymorphism (MSAP) technique has been employed on somatic embryo-derived oil palms (Elaeis guineensis Jacq.) to identify methylation polymorphisms correlated with the "mantled" somaclonal variation. The variant phenotype displays an unstable feminization of male organs in both male and female flowers. Using MSAP, the methylation status of CCGG sites was compared in three normal versus three mantled regenerants sampled in clonal populations obtained through somatic embryogenesis from four genotypically distinct mother palms. Overall, 64 selective primer combinations were used and they have amplified 23 markers exhibiting a differential methylation pattern between the two phenotypes. Our results indicate that CCGG sites are poorly affected by the considerable decrease in global DNA methylation that has been previously associated with the mantled phenotype. Each of the 23 markers isolated in the present study could discriminate between the two phenotypes only when they were from the same genetic origin. This result hampers at the moment the direct use of MSAP markers for the early detection of variants, even though valuable information on putative target sequences will be obtained from a further characterization of these polymorphic markers.

Characterization of somatic embryogenesis initiated from the Arabidopsis shoot apex.

PubMed

Kadokura, Satoshi; Sugimoto, Kaoru; Tarr, Paul; Suzuki, Takamasa; Matsunaga, Sachihiro

2018-04-28

Somatic embryogenesis is one of the best examples of the remarkable developmental plasticity of plants, in which committed somatic cells can dedifferentiate and acquire the ability to form an embryo and regenerate an entire plant. In Arabidopsis thaliana, the shoot apices of young seedlings have been reported as an alternative tissue source for somatic embryos (SEs) besides the widely studied zygotic embryos taken from siliques. Although SE induction from shoots demonstrates the plasticity of plants more clearly than the embryo-to-embryo induction system, the underlying developmental and molecular mechanisms involved are unknown. Here we characterized SE formation from shoot apex explants by establishing a system for time-lapse observation of explants during SE induction. We also established a method to distinguish SE-forming and non-SE-forming explants prior to anatomical SE formation, enabling us to identify distinct transcriptome profiles of these two explants at SE initiation. We show that embryonic fate commitment takes place at day 3 of SE induction and the SE arises directly, not through callus formation, from the base of leaf primordia just beside the shoot apical meristem (SAM), where auxin accumulates and shoot-root polarity is formed. The expression domain of a couple of key developmental genes for the SAM transiently expands at this stage. Our data demonstrate that SE-forming and non-SE-forming explants share mostly the same transcripts except for a limited number of embryonic genes and root genes that might trigger the SE-initiation program. Thus, SE-forming explants possess a mixed identity (SAM, root and embryo) at the time of SE specification. Copyright © 2018. Published by Elsevier Inc.

Will crown ideotype help determine optimum varietal silviculture?

Treesearch

Timothy J. Albaugh; Thomas R. Fox; Marco A. Yanez; Rafael A. Rubilar; Barry Goldfarb

2016-01-01

Recent advances in somatic embryogenesis permit large numbers of clonal loblolly pine (Pinus taeda L.) to be produced and deployed. Clones may have greater growth (mean annual increment exceeding 30 cubic meters per hectare per year), greater stand uniformity and may be more susceptible to genotype by environment interactions when they are deployed in intensively...

Growth and wood properties of genetically improved loblolly pine: propagation type comparison and genetic parameters

Treesearch

Finto Antony; Laurence Schimleck; Lewis Jordan; Benjamin Hornsby; Joseph Dahlen; Richard Daniels; Alexander Clark; Luis Apiolaza; Dudley Huber

2013-01-01

The use of clonal varieties in forestry offers great potential to improve growth traits (quantity) and wood properties (quality) of loblolly pine (Pinus taeda L.). Loblolly pine trees established via somatic embryogenesis (clones), full-sib zygotic crosses, and half-sib zygotic open-pollinated families were sampled to identify variation in growth and wood properties...

Intergeneric somatic hybrid plants of Citrus sinensis cv. Hamlin and Poncirus trifoliata cv. Flying Dragon.

PubMed

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

1988-01-01

Intergeneric somatic hybrid plants between 'Hamlin' sweet orange [Citrus sinensis (L.) Osbeck] and 'Flying Dragon' trifoliate orange (Poncirus trifoliata Raf.) were regenerated following protoplast fusion. 'Hamlin' protoplasts, isolated from an habituated embryogenic suspension culture, were fused chemically with 'Flying Dragon' protoplasts isolated from juvenile leaf tissue. The hybrid selection scheme was based on complementation of the regenerative ability of the 'Hamlin' protoplasts with the subsequent expression of the trifoliate leaf character of 'Flying Dragon.' Hybrid plants were regenerated via somatic embryogenesis and multiplied organogenically. Hybrid morphology was intermediate to that of the parents. Chromosome counts indicated that the hybrids were allotetraploids (2n=4x=36). Malate dehydrogenase (MDH) isozyme patterns confirmed the hybrid nature of the regenerated plants. These genetically unique somatic hybrid plants will be evaluated for citrus rootstock potential. The cell fusion, selection, and regeneration scheme developed herein should provide a general means to expand the germplasm base of cultivated Citrus by intergeneric hybridization with related sexually incompatible genera.

Genetic effects on stand-level uniformity, and above- and belowground dry mass production in juvenile loblolly pine

Treesearch

Michael J. Aspinwall; John S. King; Steven E. McKeand; Bronson P. Bullock

2012-01-01

Several decades of tree improvement operations have drastically increased loblolly pine plantation productivity in the southern U.S. (McKeand et al., 2003). This work has lead to the availability of a number of highly productive open-pollinated and full-sib families (McKeand et al., 2006). In addition, vegetative propagation (somatic embryogenesis) has also made it...

Timing, rates and spectra of human germline mutation.

PubMed

Rahbari, Raheleh; Wuster, Arthur; Lindsay, Sarah J; Hardwick, Robert J; Alexandrov, Ludmil B; Turki, Saeed Al; Dominiczak, Anna; Morris, Andrew; Porteous, David; Smith, Blair; Stratton, Michael R; Hurles, Matthew E

2016-02-01

Germline mutations are a driving force behind genome evolution and genetic disease. We investigated genome-wide mutation rates and spectra in multi-sibling families. The mutation rate increased with paternal age in all families, but the number of additional mutations per year differed by more than twofold between families. Meta-analysis of 6,570 mutations showed that germline methylation influences mutation rates. In contrast to somatic mutations, we found remarkable consistency in germline mutation spectra between the sexes and at different paternal ages. In parental germ line, 3.8% of mutations were mosaic, resulting in 1.3% of mutations being shared by siblings. The number of these shared mutations varied significantly between families. Our data suggest that the mutation rate per cell division is higher during both early embryogenesis and differentiation of primordial germ cells but is reduced substantially during post-pubertal spermatogenesis. These findings have important consequences for the recurrence risks of disorders caused by de novo mutations.

Somatic embryogenesis in wild relatives of cotton (Gossypium Spp.)

PubMed Central

Rao, Abdul Qayyum; Hussain, S. Sarfraz; Shahzad, M. Saqib; Bokhari, S. Yassir Abbas; Raza, M. Hashim; Rakha, Allah; Majeed, A.; Shahid, A. Ali; Saleem, Zafar; Husnain, Tayyab; Riazuddin, S.

2006-01-01

Wild cotton species can contribute a valuable gene pool for agronomically desirable cultivated tetraploid cultivars. In order to exploit diploid cotton a regeneration system is required to achieve transformation based goals. The present studies aimed at optimizing the conditions for regeneration of local varieties as well as wild species of cotton. Different callus induction media were tested with varying concentrations of hormones in which sucrose was used as nutritional source. Different explants (hypocotyls, cotyledon, root) were used to check the regeneration of both local cotton plants and wild relatives using T & G medium, BAP medium, CIM medium, EMMS medium, and cell suspension medium. Different stages of embryogenicity such as early torpedo stage, late torpedo stage, heart stage, globular stage and cotyledonary stage were observed in wild relatives of cotton. The results of this study pave the way for establishing future transformation methods. PMID:16532531

Germ cells are not the primary factor for sexual fate determination in goldfish.

PubMed

Goto, Rie; Saito, Taiju; Takeda, Takahiro; Fujimoto, Takafumi; Takagi, Misae; Arai, Katsutoshi; Yamaha, Etsuto

2012-10-01

The presence of germ cells in the early gonad is important for sexual fate determination and gonadal development in vertebrates. Recent studies in zebrafish and medaka have shown that a lack of germ cells in the early gonad induces sex reversal in favor of a male phenotype. However, it is uncertain whether the gonadal somatic cells or the germ cells are predominant in determining gonadal fate in other vertebrate. Here, we investigated the role of germ cells in gonadal differentiation in goldfish, a gonochoristic species that possesses an XX-XY genetic sex determination system. The primordial germ cells (PGCs) of the fish were eliminated during embryogenesis by injection of a morpholino oligonucleotide against the dead end gene. Fish without germ cells showed two types of gonadal morphology: one with an ovarian cavity; the other with seminiferous tubules. Next, we tested whether function could be restored to these empty gonads by transplantation of a single PGC into each embryo, and also determined the gonadal sex of the resulting germline chimeras. Transplantation of a single GFP-labeled PGC successfully produced a germline chimera in 42.7% of the embryos. Some of the adult germline chimeras had a developed gonad on one side that contained donor derived germ cells, while the contralateral gonad lacked any early germ cell stages. Female germline chimeras possessed a normal ovary and a germ-cell free ovary-like structure on the contralateral side; this structure was similar to those seen in female morphants. Male germline chimeras possessed a testis and a contralateral empty testis that contained some sperm in the tubular lumens. Analysis of aromatase, foxl2 and amh expression in gonads of morphants and germline chimeras suggested that somatic transdifferentiation did not occur. The offspring of fertile germline chimeras all had the donor-derived phenotype, indicating that germline replacement had occurred and that the transplanted PGC had rescued both female and male gonadal function. These findings suggest that the absence of germ cells did not affect the pathway for ovary or testis development and that phenotypic sex in goldfish is determined by somatic cells under genetic sex control rather than an interaction between the germ cells and somatic cells. Copyright © 2012 Elsevier Inc. All rights reserved.

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

PubMed

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. 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.

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

Generation of peanut mutants by fast neutron irradiation combined with in vitro culture

PubMed Central

Wang, Jing-Shan; Sui, Jiong-Ming; Xie, Yong-Dun; Guo, Hui-Jun; Qiao, Li-Xian; Zhao, Li-Lan; Yu, Shan-Lin; Liu, Lu-Xiang

2015-01-01

Induced mutations have played an important role in the development of new plant varieties. In this study, we investigated the effects of fast neutron irradiation on somatic embryogenesis combined with plant regeneration in embryonic leaflet culture to develop new peanut (Arachis hypogaea L.) germplasm for breeding. The dry seeds of the elite cultivar Luhua 11 were irradiated with fast neutrons at dosages of 9.7, 14.0 and 18.0 Gy. The embryonic leaflets were separated and incubated in a medium with 10.0-mg/l 2,4-D to induce somatic embryogenesis. Next, they were incubated in a medium with 4.0-mg/l BAP for plant regeneration. As the irradiation dosage increased, the frequency of both somatic embryo formation and plantlet regeneration decreased. The regenerated plantlets were grafted onto rootstocks and were transplanted into the field. Later, the mature seeds of the regenerated plants were harvested. The M2 generation plants from most of the regenerated cultivars exhibited variations and segregation in vigor, plant height, branch and pod number, pod size, and pod shape. To determine whether the phenotypes were associated with genomic modification, we compared the DNA polymorphisms between the wild-type plants and 19 M3-generation individuals from different regenerated plants. We used 20 pairs of simple sequence repeat (SSR) primers and detected polymorphisms between most of the mutants and the wild-type plants (Luhua 11). Our results indicate that using a combination of fast neutron irradiation and tissue culture is an effective approach for creating new peanut germplasm. PMID:25653418

Transposable elements at the center of the crossroads between embryogenesis, embryonic stem cells, reprogramming, and long non-coding RNAs.

PubMed

Hutchins, Andrew Paul; Pei, Duanqing

Transposable elements (TEs) are mobile genomic sequences of DNA capable of autonomous and non-autonomous duplication. TEs have been highly successful, and nearly half of the human genome now consists of various families of TEs. Originally thought to be non-functional, these elements have been co-opted by animal genomes to perform a variety of physiological functions ranging from TE-derived proteins acting directly in normal biological functions, to innovations in transcription factor logic and influence on epigenetic control of gene expression. During embryonic development, when the genome is epigenetically reprogrammed and DNA-demethylated, TEs are released from repression and show embryonic stage-specific expression, and in human and mouse embryos, intact TE-derived endogenous viral particles can even be detected. A similar process occurs during the reprogramming of somatic cells to pluripotent cells: When the somatic DNA is demethylated, TEs are released from repression. In embryonic stem cells (ESCs), where DNA is hypomethylated, an elaborate system of epigenetic control is employed to suppress TEs, a system that often overlaps with normal epigenetic control of ESC gene expression. Finally, many long non-coding RNAs (lncRNAs) involved in normal ESC function and those assisting or impairing reprogramming contain multiple TEs in their RNA. These TEs may act as regulatory units to recruit RNA-binding proteins and epigenetic modifiers. This review covers how TEs are interlinked with the epigenetic machinery and lncRNAs, and how these links influence each other to modulate aspects of ESCs, embryogenesis, and somatic cell reprogramming.

Transcriptome Analysis of Honeybee (Apis Mellifera) Haploid and Diploid Embryos Reveals Early Zygotic Transcription during Cleavage

PubMed Central

Pires, Camilla Valente; Freitas, Flávia Cristina de Paula; Cristino, Alexandre S.; Dearden, Peter K.; Simões, Zilá Luz Paulino

2016-01-01

In honeybees, the haplodiploid sex determination system promotes a unique embryogenesis process wherein females develop from fertilized eggs and males develop from unfertilized eggs. However, the developmental strategies of honeybees during early embryogenesis are virtually unknown. Similar to most animals, the honeybee oocytes are supplied with proteins and regulatory elements that support early embryogenesis. As the embryo develops, the zygotic genome is activated and zygotic products gradually replace the preloaded maternal material. The analysis of small RNA and mRNA libraries of mature oocytes and embryos originated from fertilized and unfertilized eggs has allowed us to explore the gene expression dynamics in the first steps of development and during the maternal-to-zygotic transition (MZT). We localized a short sequence motif identified as TAGteam motif and hypothesized to play a similar role in honeybees as in fruit flies, which includes the timing of early zygotic expression (MZT), a function sustained by the presence of the zelda ortholog, which is the main regulator of genome activation. Predicted microRNA (miRNA)-target interactions indicated that there were specific regulators of haploid and diploid embryonic development and an overlap of maternal and zygotic gene expression during the early steps of embryogenesis. Although a number of functions are highly conserved during the early steps of honeybee embryogenesis, the results showed that zygotic genome activation occurs earlier in honeybees than in Drosophila based on the presence of three primary miRNAs (pri-miRNAs) (ame-mir-375, ame-mir-34 and ame-mir-263b) during the cleavage stage in haploid and diploid embryonic development. PMID:26751956

Transcriptional profiling of epigenetic regulators in somatic embryos during temperature induced formation of an epigenetic memory in Norway spruce.

PubMed

Yakovlev, Igor A; Carneros, Elena; Lee, YeonKyeong; Olsen, Jorunn E; Fossdal, Carl Gunnar

2016-05-01

A significant number of epigenetic regulators were differentially expressed during embryogenesis at different epitype-inducing conditions. Our results support that methylation of DNA and histones, as well as sRNAs, are pivotal for the establishment of the epigenetic memory. As a forest tree species with long generation times, Norway spruce is remarkably well adapted to local environmental conditions despite having recently, from an evolutionary perspective, recolonized large areas following the last glaciation. In this species, there is an enigmatic epigenetic memory of the temperature conditions during embryogenesis that allows rapid adaptation to changing environment. We used a transcriptomic approach to investigate the molecular mechanisms underlying the formation of the epigenetic memory during somatic embryogenesis in Norway spruce. Nine mRNA libraries were prepared from three epitypes of the same genotype resulting from exposure to epitype-inducing temperatures of 18, 23 and 28 °C. RNA-Seq analysis revealed more than 10,000 differentially expressed genes (DEGs). The epitype-inducing conditions during SE were accompanied by marked transcriptomic changes for multiple gene models related to the epigenetic machinery. Out of 735 putative orthologs of epigenetic regulators, 329 were affected by the epitype-inducing temperatures and differentially expressed. The majority of DEGs among the epigenetic regulators was related to DNA and histone methylation, along with sRNA pathways and a range of putative thermosensing and signaling genes. These genes could be the main epigenetic regulators involved in formation of the epigenetic memory. We suggest considerable expansion of gene families of epigenetic regulators in Norway spruce compared to orthologous gene families in Populus and Arabidopsis. Obtained results provide a solid basis for further genome annotation and studies focusing on the importance of these candidate genes for the epigenetic memory formation.

Diverse roles of actin in C. elegans early embryogenesis

PubMed Central

Velarde, Nathalie; Gunsalus, Kristin C; Piano, Fabio

2007-01-01

Background The actin cytoskeleton plays critical roles in early development in Caenorhabditis elegans. To further understand the complex roles of actin in early embryogenesis we use RNAi and in vivo imaging of filamentous actin (F-actin) dynamics. Results Using RNAi, we found processes that are differentially sensitive to levels of actin during early embryogenesis. Mild actin depletion shows defects in cortical ruffling, pseudocleavage, and establishment of polarity, while more severe depletion shows defects in polar body extrusion, cytokinesis, chromosome segregation, and eventually, egg production. These defects indicate that actin is required for proper oocyte development, fertilization, and a wide range of important events during early embryogenesis, including proper chromosome segregation. In vivo visualization of the cortical actin cytoskeleton shows dynamics that parallel but are distinct from the previously described myosin dynamics. Two distinct types of actin organization are observed at the cortex. During asymmetric polarization to the anterior, or the establishment phase (Phase I), actin forms a meshwork of microfilaments and focal accumulations throughout the cortex, while during the anterior maintenance phase (Phase II) it undergoes a morphological transition to asymmetrically localized puncta. The proper asymmetric redistribution is dependent on the PAR proteins, while both asymmetric redistribution and morphological transitions are dependent upon PFN-1 and NMY-2. Just before cytokinesis, actin disappears from most of the cortex and is only found around the presumptive cytokinetic furrow. Finally, we describe dynamic actin-enriched comets in the early embryo. Conclusion During early C. elegans embryogenesis actin plays more roles and its organization is more dynamic than previously described. Morphological transitions of F-actin, from meshwork to puncta, as well as asymmetric redistribution, are regulated by the PAR proteins. Results from this study indicate new insights into the cellular and developmental roles of the actin cytoskeleton. PMID:18157918

LET-418/Mi2 and SPR-5/LSD1 cooperatively prevent somatic reprogramming of C. elegans germline stem cells.

PubMed

Käser-Pébernard, Stéphanie; Müller, Fritz; Wicky, Chantal

2014-04-08

Throughout their journey to forming new individuals, germline stem cells must remain totipotent, particularly by maintaining a specific chromatin structure. However, the place epigenetic factors occupy in this process remains elusive. So far, "sensitization" of chromatin by modulation of histone arrangement and/or content was believed to facilitate transcription-factor-induced germ cell reprogramming. Here, we demonstrate that the combined reduction of two epigenetic factors suffices to reprogram C. elegans germ cells. The histone H3K4 demethylase SPR-5/LSD1 and the chromatin remodeler LET-418/Mi2 function together in an early process to maintain germ cell status and act as a barrier to block precocious differentiation. This epigenetic barrier is capable of limiting COMPASS-mediated H3K4 methylation, because elevated H3K4me3 levels correlate with germ cell reprogramming in spr-5; let-418 mutants. Interestingly, germ cells deficient for spr-5 and let-418 mainly reprogram as neurons, suggesting that neuronal fate might be the first to be derepressed in early embryogenesis.

LET-418/Mi2 and SPR-5/LSD1 Cooperatively Prevent Somatic Reprogramming of C. elegans Germline Stem Cells

PubMed Central

Käser-Pébernard, Stéphanie; Müller, Fritz; Wicky, Chantal

2014-01-01

Summary Throughout their journey to forming new individuals, germline stem cells must remain totipotent, particularly by maintaining a specific chromatin structure. However, the place epigenetic factors occupy in this process remains elusive. So far, “sensitization” of chromatin by modulation of histone arrangement and/or content was believed to facilitate transcription-factor-induced germ cell reprogramming. Here, we demonstrate that the combined reduction of two epigenetic factors suffices to reprogram C. elegans germ cells. The histone H3K4 demethylase SPR-5/LSD1 and the chromatin remodeler LET-418/Mi2 function together in an early process to maintain germ cell status and act as a barrier to block precocious differentiation. This epigenetic barrier is capable of limiting COMPASS-mediated H3K4 methylation, because elevated H3K4me3 levels correlate with germ cell reprogramming in spr-5; let-418 mutants. Interestingly, germ cells deficient for spr-5 and let-418 mainly reprogram as neurons, suggesting that neuronal fate might be the first to be derepressed in early embryogenesis. PMID:24749077

A History of the Discovery of Random X Chromosome Inactivation in the Human Female and its Significance

PubMed Central

Balderman, Sophia; Lichtman, Marshall A.

2011-01-01

Genetic determinants of sex in placental mammals developed by the evolution of primordial autosomes into the male and female sex chromosomes. The Y chromosome determines maleness by the action of the gene SRY, which encodes a protein that initiates a sequence of events prompting the embryonic gonads to develop into testes. The X chromosome in the absence of a Y chromosome results in a female by permitting the conversion of the embryonic gonads into ovaries. We trace the historical progress that resulted in the discovery that one X chromosome in the female is randomly inactivated in early embryogenesis, accomplishing approximate equivalency of X chromosome gene dosage in both sexes. This event results in half of the somatic cells in a tissue containing proteins encoded by the genes of the maternal X chromosome and half having proteins encoded by the genes of the paternal X chromosome, on average, accounting for the phenotype of a female heterozygote with an X chromosome mutation. The hypothesis of X chromosome inactivation as a random event early in embryogenesis was first described as a result of studies of variegated coat color in female mice. Similar results were found in women using the X chromosome-linked gene, glucose-6-phosphate dehydrogenase, studied in red cells. The random inactivation of the X chromosome-bearing genes for isoenzyme types A and B of glucose-6-phosphate dehydrogenase was used to establish the clonal origin of neoplasms in informative women with leiomyomas. Behind these discoveries are the stories of the men and women scientists whose research enlightened these aspects of X chromosome function and their implication for medicine. PMID:23908816

Conservation of proteo-lipid nuclear membrane fusion machinery during early embryogenesis.

PubMed

Byrne, Richard D; Veeriah, Selvaraju; Applebee, Christopher J; Larijani, Banafshé

2014-01-01

The fusogenic lipid diacylglycerol is essential for remodeling gamete and zygote nuclear envelopes (NE) during early embryogenesis. It is unclear whether upstream signaling molecules are likewise conserved. Here we demonstrate PLCγ and its activator SFK1, which co-operate during male pronuclear envelope formation, also promote the subsequent male and female pronuclear fusion. PLCγ and SFK1 interact directly at the fusion site leading to PLCγ activation. This is accompanied by a spatially restricted reduction of PtdIns(4,5)P2. Consequently, pronuclear fusion is blocked by PLCγ or SFK1 inhibition. These findings identify new regulators of events in the early embryo and suggest a conserved "toolkit" of fusion machinery drives successive NE fusion events during embryogenesis.

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.

Isozyme modifications and plant regeneration through somatic embryogenesis in sweet potato (Ipomoea batatas (L.) Lam.).

PubMed

Cavalcante Alves, J M; Sihachakr, D; Allot, M; Tizroutine, S; Mussio, I; Servaes, A; Ducreux, G

1994-05-01

The potential of somatic embryogenesis was evaluated for 10 cultivars of sweet potato through extensive embryogenic response and isozyme analysis. Embryogenic callus was induced by incubating lateral buds on Murashige and Skoog medium containing 10 μM 2,4-dichlorophenoxyacetic acid for 6-8 weeks. The frequency of embryogenic response was low, and varied with genotypes, ranging from 0 to 17%. Embryo to plantlet formation could be enhanced by the use of the combination of 2,4-dichlorophenoxyacetic acid with kinetin, both used at 0.01 μM. Embryogenic callus with its potential of plantlet formation has constantly been maintained for over two years. However, after several subcultures, 0.5 to 12% of embryogenic callus reverted irreversibly into friable fast-growing non-embryogenic callus whose ability to regenerate shoots was then definitively lost. The isozymes of esterase, peroxidase, glutamate oxaloacetate transaminase and acid phosphatase investigated in this study were found appropriate to distinguish compact embryogenic from friable non-embryogenic callus in sweet potato. In fact, the callus reversion was associated with a loss of bands or a decline in isozyme activity. On the contrary, very small changes in isozyme activity or no specific changes at all were observed during the differentiation of embryogenic callus into globular embryos.

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.

5-Azacytidine combined with 2,4-D improves somatic embryogenesis of Acca sellowiana (O. Berg) Burret by means of changes in global DNA methylation levels.

PubMed

Fraga, Hugo P F; Vieira, Leila N; Caprestano, Clarissa A; Steinmacher, Douglas A; Micke, Gustavo A; Spudeit, Daniel A; Pescador, Rosete; Guerra, Miguel P

2012-12-01

DNA methylation is an epigenetic regulatory mechanism of gene expression which can be associated with developmental phases and in vitro morphogenetic competence in plants. The present work evaluated the effects of 5-azacytidine (AzaC) and 2,4-dichlorophenoxyacetic acid (2,4-D) on Acca sellowiana somatic embryogenesis (SE) and global DNA methylation levels by high-performance liquid chromatography mass spectrometry (HPLC/MS/MS). 2,4-D-free treatments revealed no somatic embryo formation in both accessions tested. Treatments supplemented with 2,4-D pulse plus AzaC in the culture medium resulted in increased embryo formation. In AzaC-free treatment, HPLC/MS/MS analysis showed a gradual increase in methylation levels in cultures of both accessions tested during SE induction. Treatment with AzaC and 2,4-D-free resulted in a marked decrease in methylation for both accessions, ranging from 37.6 to 20.8 %. In treatment with 2,4-D and AzaC combined, the 85 accession showed increasing global methylation levels. Otherwise, the 101X458 accession, in the same treatment, showed a decrease between 10 and 20 days, followed by an increase after 30 days (39.5, 36.2 and 41.6 %). These results indicate that 2,4-D pulse combined with AzaC improves SE induction. However, the conversion phase showed that although positively influencing SE induction, AzaC had a dysregulatory effect on the stage of autotrophic plant formation, resulting in significantly lower conversion rates. The results suggest that DNA methylation dramatically influences SE in Acca sellowiana, and global DNA methylation dynamics are related to morphogenetic response. 5-Azacytidine combined with 2,4-D increases the number of Acca sellowiana somatic embryos. Global DNA methylation is directly affected by these compounds.

DNA Methylation Profiling of Embryonic Stem Cell Differentiation into the Three Germ Layers

PubMed Central

Isagawa, Takayuki; Nagae, Genta; Shiraki, Nobuaki; Fujita, Takanori; Sato, Noriko; Ishikawa, Shumpei; Kume, Shoen; Aburatani, Hiroyuki

2011-01-01

Embryogenesis is tightly regulated by multiple levels of epigenetic regulation such as DNA methylation, histone modification, and chromatin remodeling. DNA methylation patterns are erased in primordial germ cells and in the interval immediately following fertilization. Subsequent developmental reprogramming occurs by de novo methylation and demethylation. Variance in DNA methylation patterns between different cell types is not well understood. Here, using methylated DNA immunoprecipitation and tiling array technology, we have comprehensively analyzed DNA methylation patterns at proximal promoter regions in mouse embryonic stem (ES) cells, ES cell-derived early germ layers (ectoderm, endoderm and mesoderm) and four adult tissues (brain, liver, skeletal muscle and sperm). Most of the methylated regions are methylated across all three germ layers and in the three adult somatic tissues. This commonly methylated gene set is enriched in germ cell-associated genes that are generally transcriptionally inactive in somatic cells. We also compared DNA methylation patterns by global mapping of histone H3 lysine 4/27 trimethylation, and found that gain of DNA methylation correlates with loss of histone H3 lysine 4 trimethylation. Our combined findings indicate that differentiation of ES cells into the three germ layers is accompanied by an increased number of commonly methylated DNA regions and that these tissue-specific alterations in methylation occur for only a small number of genes. DNA methylation at the proximal promoter regions of commonly methylated genes thus appears to be an irreversible mark which functions to fix somatic lineage by repressing the transcription of germ cell-specific genes. PMID:22016810

DNA methylation profiling of embryonic stem cell differentiation into the three germ layers.

PubMed

Isagawa, Takayuki; Nagae, Genta; Shiraki, Nobuaki; Fujita, Takanori; Sato, Noriko; Ishikawa, Shumpei; Kume, Shoen; Aburatani, Hiroyuki

2011-01-01

Embryogenesis is tightly regulated by multiple levels of epigenetic regulation such as DNA methylation, histone modification, and chromatin remodeling. DNA methylation patterns are erased in primordial germ cells and in the interval immediately following fertilization. Subsequent developmental reprogramming occurs by de novo methylation and demethylation. Variance in DNA methylation patterns between different cell types is not well understood. Here, using methylated DNA immunoprecipitation and tiling array technology, we have comprehensively analyzed DNA methylation patterns at proximal promoter regions in mouse embryonic stem (ES) cells, ES cell-derived early germ layers (ectoderm, endoderm and mesoderm) and four adult tissues (brain, liver, skeletal muscle and sperm). Most of the methylated regions are methylated across all three germ layers and in the three adult somatic tissues. This commonly methylated gene set is enriched in germ cell-associated genes that are generally transcriptionally inactive in somatic cells. We also compared DNA methylation patterns by global mapping of histone H3 lysine 4/27 trimethylation, and found that gain of DNA methylation correlates with loss of histone H3 lysine 4 trimethylation. Our combined findings indicate that differentiation of ES cells into the three germ layers is accompanied by an increased number of commonly methylated DNA regions and that these tissue-specific alterations in methylation occur for only a small number of genes. DNA methylation at the proximal promoter regions of commonly methylated genes thus appears to be an irreversible mark which functions to fix somatic lineage by repressing the transcription of germ cell-specific genes.

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.

Reproductive effects of the water-accommodated fraction of a natural gas condensate in the Indo-Pacific reef-building coral Pocillopora damicornis.

PubMed

Villanueva, R D; Yap, H T; Montaño, M N E

2011-11-01

Toxic effects of the water-accommodated fraction (WAF) of a natural gas condensate on the reproduction of the brooding coral Pocillopora damicornis were studied in short-term (24 h) laboratory experiments. Coral fragments were exposed to varying concentrations of condensate WAF during different reproductive phases: gametogenesis, early embryogenesis, and late embryogenesis (when nighttime planulation occurs). During gametogenesis, exposure to condensate WAF did not inhibit subsequent production of larvae. On the other hand, exposure to >25% WAF of gravid corals, at early and late embryogenesis, resulted in abortion and early release of larvae, respectively, with higher percentages of larvae expelled in fragments treated with higher concentrations of condensate WAF at least 3h after onset of exposure. Aborted larvae during early embryogenesis were 'premature', as they are of small size (0.06±0.03 mm³), low metamorphic competency (54%), and white in coloration, with a pale brown oral end (indicating low density of zooxanthellae). Those larvae released at the latter part of embryogenesis are bigger in size (0.22±0.08 mm³), possess 100% metamorphic competency, and are brown in coloration (high density of zooxanthellae). Aside from direct effects on reproduction, fragment mortality index was higher in samples exposed to higher concentrations of condensate WAF (>25%), hence lowering the number of potentially reproducing polyps. Altogether, exposure to >25% natural gas condensate WAF for at least 3h can potentially disrupt the replenishment of coral populations due to negative effects on reproduction and early life processes. Copyright © 2011 Elsevier Inc. All rights reserved.

Enhanced Indirect Somatic Embryogenesis of Date Palm Using Low Levels of Seawater.

PubMed

Taha, Rania A

2017-01-01

Date palm tolerates salinity, drought, and high temperatures. Arid and semiarid zones, especially the Middle East region, need a huge number of date palms for cultivation. To meet this demand, tissue culture techniques have great potential for mass production of plantlets, especially using the indirect embryogenesis technique; any improvement of these techniques is a worthy objective. Low levels of salinity can enhance growth and development of tolerant plants. A low level of seawater, a natural source of salinity, reduces the time required for micropropagation processes of date palm cv. Malkaby when added to MS medium. Medium containing seawater at 500 ppm total dissolved solid (TDS) (12.2 mL/L) improves callus proliferation, whereas 1500 ppm (36.59 mL/L) enhances plant regeneration including multiplication of secondary embryos, embryo germination, and rooting.

Elimination of Grapevine leafroll associated virus-3, Grapevine rupestris stem pitting associated virus and Grapevine virus A from a Tunisian Cultivar by Somatic Embryogenesis and Characterization of the Somaclones Using Ampelographic Descriptors

PubMed Central

Bouamama-Gzara, Badra; Selmi, Ilhem; Chebil, Samir; Melki, Imene; Mliki, Ahmed; Ghorbel, Abdelwahed; Carra, Angela; Carimi, Francesco; Mahfoudhi, Naima

2017-01-01

Prospecting of local grapevine (Vitis vinifera L.) germplasm revealed that Tunisia possesses a rich patrimony which presents diversified organoleptic characteristics. However, viral diseases seriously affect all local grapevine cultivars which risk a complete extinction. Sanitation programs need to be established to preserve and exploit, as a gene pool, the Tunisian vineyards areas. The presence of the Grapevine leafroll associated virus-3 (GLRaV-3), Grapevine stem pitting associated virus (GRSPaV) and Grapevine virus A (GVA), were confirmed in a Tunisian grapevine cultivar using serological and molecular analyses. The association between GRSPaV and GVA viruses induces more rugose wood symptoms and damages. For this reason the cleansing of the infected cultivar is highly advisable. Direct and recurrent somatic embryos of cv. ‘Hencha’ were successfully induced from filament, when cultured on Chée and Pool (1987). based-medium, enriched with 2 mg 1−1 of 2,4-dichlorophenoxyacetic acid and 2.5 mg 1−1 of Thidiazuron, after 36 weeks of culture. After six months of acclimatization, RT-PCR carried on 50 somaplants confirmed the absence of GVA, GRSPa-V as well as GLRaV-3 viruses in all somaplants. Ampelographic analysis, based on eight OIV descriptors, was carried out on two years acclimated somaplants, compared to the mother plant. Results demonstrated that the shape and contours of 46 somaclones leaves are identical to mother plant leaves and four phenotypically off-type plants were observed. The healthy state of 100% ‘Hencha’ somaclones and the high percentage of phenotypically true-to-type plants demonstrate that somatic embryogenesis is a promising technique to adopt for grapevine viruses elimination. PMID:29238279

Elimination of Grapevine leafroll associated virus-3, Grapevine rupestris stem pitting associated virus and Grapevine virus A from a Tunisian Cultivar by Somatic Embryogenesis and Characterization of the Somaclones Using Ampelographic Descriptors.

PubMed

Bouamama-Gzara, Badra; Selmi, Ilhem; Chebil, Samir; Melki, Imene; Mliki, Ahmed; Ghorbel, Abdelwahed; Carra, Angela; Carimi, Francesco; Mahfoudhi, Naima

2017-12-01

Prospecting of local grapevine ( Vitis vinifera L.) germplasm revealed that Tunisia possesses a rich patrimony which presents diversified organoleptic characteristics. However, viral diseases seriously affect all local grapevine cultivars which risk a complete extinction. Sanitation programs need to be established to preserve and exploit, as a gene pool, the Tunisian vineyards areas. The presence of the Grapevine leafroll associated virus-3 (GLRaV-3), Grapevine stem pitting associated virus (GRSPaV) and Grapevine virus A (GVA), were confirmed in a Tunisian grapevine cultivar using serological and molecular analyses. The association between GRSPaV and GVA viruses induces more rugose wood symptoms and damages. For this reason the cleansing of the infected cultivar is highly advisable. Direct and recurrent somatic embryos of cv. 'Hencha' were successfully induced from filament, when cultured on Chée and Pool (1987). based-medium, enriched with 2 mg 1 -1 of 2,4-dichlorophenoxyacetic acid and 2.5 mg 1 -1 of Thidiazuron, after 36 weeks of culture. After six months of acclimatization, RT-PCR carried on 50 somaplants confirmed the absence of GVA, GRSPa-V as well as GLRaV-3 viruses in all somaplants. Ampelographic analysis, based on eight OIV descriptors, was carried out on two years acclimated somaplants, compared to the mother plant. Results demonstrated that the shape and contours of 46 somaclones leaves are identical to mother plant leaves and four phenotypically off-type plants were observed. The healthy state of 100% 'Hencha' somaclones and the high percentage of phenotypically true-to-type plants demonstrate that somatic embryogenesis is a promising technique to adopt for grapevine viruses elimination.

Stable transformation via particle bombardment in two different soybean regeneration systems.

PubMed

Sato, S; Newell, C; Kolacz, K; Tredo, L; Finer, J; Hinchee, M

1993-05-01

The Biolistics(®) particle delivery system for the transformation of soybean (Glycine max L. Merr.) was evaluated in two different regeneration systems. The first system was multiple shoot proliferation from shoot tips obtained from immature zygotic embryos of the cultivar Williams 82, and the second was somatic embryogenesis from a long term proliferative suspension culture of the cultivar Fayette. Bombardment of shoot tips with tungsten particles, coated with precipitated DNA containing the gene for β-glucuronidase (GUS), produced GUS-positive sectors in 30% of the regenerated shoots. However, none of the regenerants which developed into plants continued to produce GUS positive tissue. Bombardment of embryogenic suspension cultures produced GUS positive globular somatic embryos which proliferated into GUS positive somatic embryos and plants. An average of 4 independent transgenic lines were generated per bombarded flask of an embryogenic suspension. Particle bombardment delivered particles into the first two cell layers of either shoot tips or somatic embryos. Histological analysis indicated that shoot organogenesis appeared to involve more than the first two superficial cell layers of a shoot tip, while somatic embryo proliferation occurred from the first cell layer of existing somatic embryos. The different transformation results obtained with these two systems appeared to be directly related to differences in the cell types which were responsible for regeneration and their accessibility to particle penetration.

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.

Mobile genetic elements and cancer. From mutations to gene therapy.

PubMed

Kozeretska, I A; Demydov, S V; Ostapchenko, L I

2011-12-01

In the present review, an association between cancer and the activity of the non-LTR retroelements L1, Alu, and SVA, as well as endogenous retroviruses, in the human genome, is analyzed. Data suggesting that transposons have been involved in embryogenesis and malignization processes, are presented. Events that lead to the activation of mobile elements in mammalian somatic cells, as well as the use of mobile elements in genetic screening and cancer gene therapy, are reviewed.

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

Progress of tissue culture and genetic transformation research in pigeon pea [Cajanus cajan (L.) Millsp.].

PubMed

Krishna, Gaurav; Reddy, P Sairam; Ramteke, P W; Bhattacharya, P S

2010-10-01

Pigeon pea [Cajanus cajan (L.) Millsp.] (Family: Fabaceae) is an important legume crop cultivated across 50 countries in Asia, Africa, and the Americas; and ranks fifth in area among pulses after soybean, common bean, peanut, and chickpea. It is consumed as a major source of protein (21%) to the human population in many developing countries. In India, it is the second important food legume contributing to 80% of the global production. Several biotic and abiotic stresses are posing a big threat to its production and productivity. Attempts to address these problems through conventional breeding methods have met with partial success. This paper reviews the chronological progress made in tissue culture through organogenesis and somatic embryogenesis, including the influence of factors such as genotypes, explant sources, and culture media including the supplementation of plant growth regulators. Comprehensive lists of morphogenetic pathways involved in in vitro regeneration through organogenesis and somatic embryogenesis using different explant tissues of diverse pigeon pea genotypes are presented. Similarly, the establishment of protocols for the production of transgenics via particle bombardment and Agrobacterium-mediated transformation using different explant tissues, Agrobacterium strains, Ti plasmids, and plant selectable markers, as well as their interactions on transformation efficiency have been discussed. Future research thrusts on the use of different promoters and stacking of genes for various biotic and abiotic stresses in pigeon pea are suggested.

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

PubMed

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

2015-03-06

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.

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

Quantification of Anti-Addictive Alkaloids Ibogaine and Voacangine in In Vivo- and In Vitro-Grown Plants of Two Mexican Tabernaemontana Species.

PubMed

Krengel, Felix; Herrera Santoyo, Josefina; Olivera Flores, Teresa de Jesús; Chávez Ávila, Víctor M; Pérez Flores, Francisco J; Reyes Chilpa, Ricardo

2016-12-01

Tabernaemontana alba and Tabernaemontana arborea are Apocynaceae species used in Mexican traditional medicine for which little phytochemical information exists. In this study, preliminary gas chromatography/mass spectrometry analyses of different organs obtained from wild plants of both species identified a total of 10 monoterpenoid indole alkaloids (MIAs) and one simple indole alkaloid, nine of which were reported for the first time in these species. Furthermore, callus cultures were established from T. alba leaf explants and regeneration of whole plants was accomplished via somatic embryogenesis. The anti-addictive MIAs ibogaine and voacangine were then quantified by gas chromatography with flame ionization detection in wild plants of both species, as well as greenhouse-grown plants, in vitro-grown plantlets and embryogenic callus of T. alba. Ibogaine and voacangine were present in most samples taken from the whole plants of both species, with stem and root barks showing the highest concentrations. No alkaloids were detected in callus samples. It was concluded that T. alba and T. arborea are potentially viable sources of ibogaine and voacangine, and that these MIAs can be produced through somatic embryogenesis and whole plant regeneration of T. alba. Approaches to increase MIA yields in whole plants and to achieve alkaloid production directly in cell cultures are discussed. © 2016 Wiley-VHCA AG, Zurich, Switzerland.

Autosomal dominant polycystic kidney disease caused by somatic and germline mosaicism.

PubMed

Tan, A Y; Blumenfeld, J; Michaeel, A; Donahue, S; Bobb, W; Parker, T; Levine, D; Rennert, H

2015-04-01

Autosomal dominant polycystic kidney disease (ADPKD) is a heterogeneous genetic disorder caused by loss of function mutations of PKD1 or PKD2 genes. Although PKD1 is highly polymorphic and the new mutation rate is relatively high, the role of mosaicism is incompletely defined. Herein, we describe the molecular analysis of ADPKD in a 19-year-old female proband and her father. The proband had a PKD1 truncation mutation c.10745dupC (p.Val3584ArgfsX43), which was absent in paternal peripheral blood lymphocytes (PBL). However, very low quantities of this mutation were detected in the father's sperm DNA, but not in DNA from his buccal cells or urine sediment. Next generation sequencing (NGS) analysis determined the level of this mutation in the father's PBL, buccal cells and sperm to be ∼3%, 4.5% and 10%, respectively, consistent with somatic and germline mosaicism. The PKD1 mutation in ∼10% of her father's sperm indicates that it probably occurred early in embryogenesis. In ADPKD cases where a de novo mutation is suspected because of negative PKD gene testing of PBL, additional evaluation with more sensitive methods (e.g. NGS) of the proband PBL and paternal sperm can enhance detection of mosaicism and facilitate genetic counseling. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Differential expression of conserved germ line markers and delayed segregation of male and female primordial germ cells in a hermaphrodite, the leech helobdella.

PubMed

Cho, Sung-Jin; Vallès, Yvonne; Weisblat, David A

2014-02-01

In sexually reproducing animals, primordial germ cells (PGCs) are often set aside early in embryogenesis, a strategy that minimizes the risk of genomic damage associated with replication and mitosis during the cell cycle. Here, we have used germ line markers (piwi, vasa, and nanos) and microinjected cell lineage tracers to show that PGC specification in the leech genus Helobdella follows a different scenario: in this hermaphrodite, the male and female PGCs segregate from somatic lineages only after more than 20 rounds of zygotic mitosis; the male and female PGCs share the same (mesodermal) cell lineage for 19 rounds of zygotic mitosis. Moreover, while all three markers are expressed in both male and female reproductive tissues of the adult, they are expressed differentially between the male and female PGCs of the developing embryo: piwi and vasa are expressed preferentially in female PGCs at a time when nanos is expressed preferentially in male PGCs. A priori, the delayed segregation of male and female PGCs from somatic tissues and from one another increases the probability of mutations affecting both male and female PGCs of a given individual. We speculate that this suite of features, combined with a capacity for self-fertilization, may contribute to the dramatically rearranged genome of Helobdella robusta relative to other animals.

Differential Expression of Conserved Germ Line Markers and Delayed Segregation of Male and Female Primordial Germ Cells in a Hermaphrodite, the Leech Helobdella

PubMed Central

Cho, Sung-Jin; Vallès, Yvonne; Weisblat, David A.

2014-01-01

In sexually reproducing animals, primordial germ cells (PGCs) are often set aside early in embryogenesis, a strategy that minimizes the risk of genomic damage associated with replication and mitosis during the cell cycle. Here, we have used germ line markers (piwi, vasa, and nanos) and microinjected cell lineage tracers to show that PGC specification in the leech genus Helobdella follows a different scenario: in this hermaphrodite, the male and female PGCs segregate from somatic lineages only after more than 20 rounds of zygotic mitosis; the male and female PGCs share the same (mesodermal) cell lineage for 19 rounds of zygotic mitosis. Moreover, while all three markers are expressed in both male and female reproductive tissues of the adult, they are expressed differentially between the male and female PGCs of the developing embryo: piwi and vasa are expressed preferentially in female PGCs at a time when nanos is expressed preferentially in male PGCs. A priori, the delayed segregation of male and female PGCs from somatic tissues and from one another increases the probability of mutations affecting both male and female PGCs of a given individual. We speculate that this suite of features, combined with a capacity for self-fertilization, may contribute to the dramatically rearranged genome of Helobdella robusta relative to other animals. PMID:24217283

Role of Auxin in Orchid Development

PubMed Central

Novak, Stacey D.; Luna, Lila J.; Gamage, Roshan N.

2014-01-01

Auxin's capacity to regulate aspects of plant development has been well characterized in model plant systems. In contrast, orchids have received considerably less attention, but the realization that many orchid species are endangered has led to culture-based propagation studies which have unveiled some functions for auxin in this system. This mini-review summarizes the many auxin-mediated developmental responses in orchids that are consistent with model systems; however, it also brings to the forefront auxin responses that are unique to orchid development, namely protocorm formation and ovary/ovule maturation. With regard to shoot establishment, we also assess auxin's involvement in orchid germination, PLB formation, and somatic embryogenesis. Further, it makes evident that auxin flow during germination of the undifferentiated, but mature, orchid embryo mirrors late embryogenesis of typical angiosperms. Also discussed is the use of orchid protocorms in future phytohormone studies to better understand the mechanisms behind meristem formation and organogenesis. PMID:25482818

Refining the application of direct embryogenesis in sugarcane: Effect of the developmental phase of leaf disc explants and the timing of DNA transfer on transformation efficiency.

PubMed

Snyman, S J; Meyer, G M; Richards, J M; Haricharan, N; Ramgareeb, S; Huckett, B I

2006-10-01

A rapid in vitro protocol using direct somatic embryogenesis and microprojectile bombardment was investigated to establish the developmental phases most suitable for efficient sugarcane transformation. Immature leaf roll disc explants with and without pre-emergent inflorescence tissue were compared. It was shown that for effective transformation to occur, explants should be cultured for several days to allow initiation of embryo development prior to bombardment. Leaf roll discs with pre-emergent inflorescences showed a higher degree of embryogenic competence than non-flowering explants, and transformation efficiency was higher when explants containing floral initials were bombarded. Despite the occurrence of high numbers of phenotypically negative plants, combining the use of inflorescent leaf roll discs with direct embryogenic regeneration has the potential to improve the speed and efficiency of transgenesis in sugarcane.

Next generation sequencing as a useful tool in the diagnostics of mosaicism in Alport syndrome.

PubMed

Beicht, Sonja; Strobl-Wildemann, Gertrud; Rath, Sabine; Wachter, Oliver; Alberer, Martin; Kaminsky, Elke; Weber, Lutz T; Hinrichsen, Tanja; Klein, Hanns-Georg; Hoefele, Julia

2013-09-10

Alport syndrome (ATS) is a progressive hereditary nephropathy characterized by hematuria and/or proteinuria with structural defects of the glomerular basement membrane. It can be associated with extrarenal manifestations (high-tone sensorineural hearing loss and ocular abnormalities). Somatic mutations in COL4A5 (X-linked), COL4A3 and COL4A4 genes (both autosomal recessive and autosomal dominant) cause Alport syndrome. Somatic mosaicism in Alport patients is very rare. The reason for this may be due to the difficulty of detection. We report the case of a boy and his mother who presented with Alport syndrome. Mutational analysis showed the novel hemizygote pathogenic mutation c.2396-1G>A (IVS29-1G>A) at the splice acceptor site of the intron 29 exon 30 boundary of the COL4A5 gene in the boy. The mutation in the mother would not have been detected by Sanger sequencing without the knowledge of the mutational analysis result of her son. Further investigation of the mother using next generation sequencing showed somatic mosaicism and implied potential germ cell mosaicism. The mutation in the mother has most likely occurred during early embryogenesis. Analysis of tissue of different embryonic origin in the mother confirmed mosaicism in both mesoderm and ectoderm. Low grade mosaicism is very difficult to detect by Sanger sequencing. Next generation sequencing is increasingly used in the diagnostics and might improve the detection of mosaicism. In the case of definite clinical symptoms of ATS and missing detection of a mutation by Sanger sequencing, mutational analysis should be performed by next generation sequencing. Copyright © 2013 Elsevier B.V. All rights reserved.

Notch and Delta mRNAs in early-stage and mid-stage Drosophila embryos exhibit complementary patterns of protein producing potentials

PubMed Central

Shepherd, Andrew; Wesley, Uma; Wesley, Cedric

2010-01-01

Notch and Delta proteins generate Notch signaling that specifies cell fates during animal development. There is an intriguing phenomenon in Drosophila embryogenesis that has not received much attention and whose significance to embryogenesis is unknown. Notch and Delta mRNAs expressed in early-stage embryos are shorter than their counterparts in mid-stage embryos. We show here that the difference in sizes is due to mRNA 3′ processing at alternate polyadenylation sites. While the early-stage Notch mRNA has a lower protein-producing potential than the mid-stage Notch mRNA, the early-stage Delta mRNA has a higher protein-producing potential than the mid-stage Delta mRNA. Our data can explain the complementary patterns of Notch and Delta protein levels in early-stage and mid-stage embryos. Our data also raise the possibility that the manner and regulation of Notch signaling change in the course of embryogenesis and that this change is effected by 3′ UTR and mRNA 3′ processing factors. PMID:20201103

Apo-14 is required for digestive system organogenesis during fish embryogenesis and larval development.

PubMed

Xia, Jian-Hong; Liu, Jing-Xia; Zhou, Li; Li, Zhi; Gui, Jian-Fang

2008-01-01

Apo-14 is a fish-specific apolipoprotein and its biological function remains unknown. In this study, CagApo-14 was cloned from gibel carp (Carassius auratus gibelio) and its expression pattern was investigated during embryogenesis and early larval development. The CagApo-14 transcript and its protein product were firstly localized in the yolk syncytial layer at a high level during embryogenesis, and then found to be restricted to the digestive system including liver and intestine in later embryos and early larvae. Immunofluorescence staining in larvae and adults indicated that Cag Apo-14 protein was predominantly synthesized in and excreted from sinusoidal endothelial cells of liver tissue. Morpholino knockdown of Cag Apo-14 resulted in severe disruption of digestive organs including liver, intestine, pancreas and swim bladder. Moreover, yolk lipid transportation and utilization were severely affected in the Cag Apo-14 morphants. Overall, this data indicates that Cag Apo-14 is required for digestive system organogenesis during fish embryogenesis and larval development.

Regulation of germ cell development by intercellular signaling in the mammalian ovarian follicle.

PubMed

Clarke, Hugh J

2018-01-01

Prior to ovulation, the mammalian oocyte undergoes a process of differentiation within the ovarian follicle that confers on it the ability to give rise to an embryo. Differentiation comprises two phases-growth, during which the oocyte increases more than 100-fold in volume as it accumulates macromolecules and organelles that will sustain early embryogenesis; and meiotic maturation, during which the oocyte executes the first meiotic division and prepares for the second division. Entry of an oocyte into the growth phase appears to be triggered when the adjacent granulosa cells produce specific growth factors. As the oocyte grows, it elaborates a thick extracellular coat termed the zona pellucida. Nonetheless, cytoplasmic extensions of the adjacent granulosa cells, termed transzonal projections (TZPs), enable them to maintain contact-dependent communication with the oocyte. Through gap junctions located where the TZP tips meet the oocyte membrane, they provide the oocyte with products that sustain its metabolic activity and signals that regulate its differentiation. Conversely, the oocyte secretes diffusible growth factors that regulate proliferation and differentiation of the granulosa cells. Gap junction-permeable products of the granulosa cells prevent precocious initiation of meiotic maturation, and the gap junctions also enable oocyte maturation to begin in response to hormonal signals received by the granulosa cells. Development of the oocyte or the somatic compartment may also be regulated by extracellular vesicles newly identified in follicular fluid and at TZP tips, which could mediate intercellular transfer of macromolecules. Oocyte differentiation thus depends on continuous signaling interactions with the somatic cells of the follicle. WIREs Dev Biol 2018, 7:e294. doi: 10.1002/wdev.294 This article is categorized under: Gene Expression and Transcriptional Hierarchies > Cellular Differentiation Signaling Pathways > Cell Fate Signaling Early Embryonic Development > Gametogenesis. © 2017 Wiley Periodicals, Inc.

In vitro propagation of carnation (Dianthus caryophyllus L.).

PubMed

Casas, Jose L; Olmos, Enrique; Piqueras, Abel

2010-01-01

Carnation (Dianthus caryophyllus L.) is one of the most popular ornamental plants worldwide and also among the most studied ones, mainly in cut flower postharvest physiology. Several protocols for the in vitro propagation of this species including nodal segment culture, somatic embryogenesis, and adventitious shoot induction are described in this chapter. The presence of hyperhydricity as an abnormality during micropropagation of carnation plants has also been the object of research for many years and different strategies to overcome this problem are also included in this study.

Extrinsic Embryonic Sensory Stimulation Alters Multimodal Behavior and Cellular Activation

PubMed Central

Markham, Rebecca G.; Shimizu, Toru; Lickliter, Robert

2009-01-01

Embryonic vision is generated and maintained by spontaneous neuronal activation patterns, yet extrinsic stimulation also sculpts sensory development. Because the sensory and motor systems are interconnected in embryogenesis, how extrinsic sensory activation guides multimodal differentiation is an important topic. Further, it is unknown whether extrinsic stimulation experienced near sensory sensitivity onset contributes to persistent brain changes, ultimately affecting postnatal behavior. To determine the effects of extrinsic stimulation on multimodal development, we delivered auditory stimulation to bobwhite quail groups during early, middle, or late embryogenesis, and then tested postnatal behavioral responsiveness to auditory or visual cues. Auditory preference tendencies were more consistently toward the conspecific stimulus for animals stimulated during late embryogenesis. Groups stimulated during middle or late embryogenesis showed altered postnatal species-typical visual responsiveness, demonstrating a persistent multimodal effect. We also examined whether auditory-related brain regions are receptive to extrinsic input during middle embryogenesis by measuring postnatal cellular activation. Stimulated birds showed a greater number of ZENK-immunopositive cells per unit volume of brain tissue in deep optic tectum, a midbrain region strongly implicated in multimodal function. We observed similar results in the medial and caudomedial nidopallia in the telencephalon. There were no ZENK differences between groups in inferior colliculus or in caudolateral nidopallium, avian analog to prefrontal cortex. To our knowledge, these are the first results linking extrinsic stimulation delivered so early in embryogenesis to changes in postnatal multimodal behavior and cellular activation. The potential role of competitive interactions between the sensory and motor systems is discussed. PMID:18777564

Propagation and conservation of native forest genetic resources of medicinal use by means of in vitro and ex vitro techniques.

PubMed

Sharry, Sandra; Adema, Marina; Basiglio Cordal, María A; Villarreal, Blanca; Nikoloff, Noelia; Briones, Valentina; Abedini, Walter

2011-07-01

In Argentina, there are numerous native species which are an important source of natural products and which are traditionally used in medicinal applications. Some of these species are going through an intense extraction process in their natural habitat which may affect their genetic diversity. The aim of this study was to establish vegetative propagation systems for three native forestal species of medicinal interest. This will allow the rapid obtainment of plants to preserve the germplasm. This study included the following species which are widely used in folk medicine and its applications: Erythrina crista-galli or "seibo" (astringent, used for its cicatrizant properties and for bronchiolitic problems); Acacia caven or "espinillo" (antirheumatic, digestive, diuretic and with cicatrizant properties) and Salix humboldtiana or "sauce criollo" (antipyretic, sedative, antispasmodic, astringent). The methodology included the micropropagation of seibo, macro and micropropagation of Salix humboldtiana and the somatic embryogenesis of Acacia caven. The protocol for seibo regeneration was adjusted from nodal sections of seedlings which were obtained from seeds germinated in vitro. The macropropagation through rooted cuttings of "sauce criollo" was achieved and complete plants of this same species were obtained through both direct and indirect organogenesis using in vitro cultures. The somatic embryogenesis for Acacia caven was optimized and this led to obtain a high percentage of embryos in different stages of development. We are able to support the conservation of native forest resources of medicinal use by means of vegetative propagation techniques.

Repeated Measurements on Distinct Scales With Censoring—A Bayesian Approach Applied to Microarray Analysis of Maize

PubMed Central

Love, Tanzy; Carriquiry, Alicia

2009-01-01

We analyze data collected in a somatic embryogenesis experiment carried out on Zea mays at Iowa State University. The main objective of the study was to identify the set of genes in maize that actively participate in embryo development. Embryo tissue was sampled and analyzed at various time periods and under different mediums and light conditions. As is the case in many microarray experiments, the operator scanned each slide multiple times to find the slide-specific ‘optimal’ laser and sensor settings. The multiple readings of each slide are repeated measurements on different scales with differing censoring; they cannot be considered to be replicate measurements in the traditional sense. Yet it has been shown that the choice of reading can have an impact on genetic inference. We propose a hierarchical modeling approach to estimating gene expression that combines all available readings on each spot and accounts for censoring in the observed values. We assess the statistical properties of the proposed expression estimates using a simulation experiment. As expected, combining all available scans using an approach with good statistical properties results in expression estimates with noticeably lower bias and root mean squared error relative to other approaches that have been proposed in the literature. Inferences drawn from the somatic embryogenesis experiment, which motivated this work changed drastically when data were analyzed using the standard approaches or using the methodology we propose. PMID:19960120

LONO1 Encoding a Nucleoporin Is Required for Embryogenesis and Seed Viability in Arabidopsis1[C][W][OA

PubMed Central

Braud, Christopher; Zheng, Wenguang; Xiao, Wenyan

2012-01-01

Early embryogenesis in Arabidopsis (Arabidopsis thaliana) is distinguished by a predictable pattern of cell divisions and is a good system for investigating mechanisms of developmental pattern formation. Here, we identified a gene called LONO1 (LNO1) in Arabidopsis in which mutations can abolish the first asymmetrical cell division of the zygote, alter planes and number of cell divisions in early embryogenesis, and eventually arrest embryo development. LNO1 is highly expressed in anthers of flower buds, stigma papilla of open flowers, and embryo and endosperm during early embryogenesis, which is correlated with its functions in reproductive development. The homozygous lno1-1 seed is not viable. LNO1, a homolog of the nucleoporin NUP214 in human (Homo sapiens) and Nup159 in yeast (Saccharomyces cerevisiae), encodes a nucleoporin protein containing phenylalanine-glycine repeats in Arabidopsis. We demonstrate that LNO1 can functionally complement the defect in the yeast temperature-sensitive nucleoporin mutant nup159. We show that LNO1 specifically interacts with the Arabidopsis DEAD-box helicase/ATPase LOS4 in the yeast two-hybrid assay. Furthermore, mutations in AtGLE1, an Arabidopsis homolog of the yeast Gle1 involved in the same poly(A) mRNA export pathway as Nup159, also result in seed abortion. Our results suggest that LNO1 is a component of the nuclear pore complex required for mature mRNA export from the nucleus to the cytoplasm, which makes LNO1 essential for embryogenesis and seed viability in Arabidopsis. PMID:22898497

Dynamics of post-translationally modified histones during barley pollen embryogenesis in the presence or absence of the epi-drug trichostatin A.

PubMed

Pandey, Pooja; Daghma, Diaa S; Houben, Andreas; Kumlehn, Jochen; Melzer, Michael; Rutten, Twan

2017-06-01

Improving pollen embryogenesis. Despite the agro-economic importance of pollen embryogenesis, the mechanisms underlying this process are still poorly understood. We describe the dynamics of chromatin modifications (histones H3K4me2, H3K9ac, H3K9me2, and H3K27me3) and chromatin marks (RNA polymerase II CDC phospho-Ser5, and CENH3) during barley pollen embryogenesis. Immunolabeling results show that, in reaction to stress, immature pollen rapidly starts reorganizing several important chromatin modifications indicative of a change in cell fate. This new chromatin modification pattern was accomplished within 24 h from whereon it remained unaltered during subsequent mitotic activity. This indicates that cell fate transition, the central element of pollen embryogenesis, is completed early on during the induction process. Application of the histone deacetylase inhibitor trichostatin A stimulated pollen embryogenesis when used on pollen with a gametophytic style chromatin pattern. However, when this drug was administered to embryogenic pollen, the chromatin markers reversed toward a gametophytic profile, embryogenesis was halted and all pollen invariably died.

Expression of the homeotic gene mab-5 during Caenorhabditis elegans embryogenesis.

PubMed

Cowing, D W; Kenyon, C

1992-10-01

mab-5 is a member of a complex of homeobox-containing genes evolutionarily related to the Antennapedia and bithorax complexes of Drosophila melanogaster. Like the homeotic genes in Drosophila, mab-5 is required in a particular region along the anterior-posterior body axis, and acts during postembryonic development to give cells in this region their characteristic identities. We have used a mab-5-lacZ fusion integrated into the C. elegans genome to study the posterior-specific expression of mab-5 during embryogenesis. The mab-5-lacZ fusion was expressed in the posterior of the embryo by 180 minutes after the first cleavage, indicating that the mechanisms responsible for the position-specific expression of mab-5-lacZ act at a relatively early stage of embryogenesis. In embryos homozygous for mutations in the par genes, which disrupt segregation of factors during early cleavages, expression of mab-5-lacZ was no longer localized to the posterior. This suggests that posterior-specific expression of mab-5 depends on the appropriate segregation of developmental factors during early embryogenesis. After extrusion of any blastomere of the four-cell embryo, descendants of the remaining three cells could still express the mab-5-lacZ fusion. In these partial embryos, however, the fusion was often expressed in cells scattered throughout the embryo, suggesting that cell-cell interactions and/or proper positioning of early blastomeres are required for mab-5 expression to be localized to the posterior.

Engineered LINE-1 retrotransposition in nondividing human neurons

PubMed Central

Macia, Angela; Widmann, Thomas J.; Heras, Sara R.; Ayllon, Veronica; Sanchez, Laura; Benkaddour-Boumzaouad, Meriem; Muñoz-Lopez, Martin; Rubio, Alejandro; Amador-Cubero, Suyapa; Blanco-Jimenez, Eva; Garcia-Castro, Javier; Menendez, Pablo; Ng, Philip; Muotri, Alysson R.; Goodier, John L.; Garcia-Perez, Jose L.

2017-01-01

Half the human genome is made of transposable elements (TEs), whose ongoing activity continues to impact our genome. LINE-1 (or L1) is an autonomous non-LTR retrotransposon in the human genome, comprising 17% of its genomic mass and containing an average of 80–100 active L1s per average genome that provide a source of inter-individual variation. New LINE-1 insertions are thought to accumulate mostly during human embryogenesis. Surprisingly, the activity of L1s can further impact the somatic human brain genome. However, it is currently unknown whether L1 can retrotranspose in other somatic healthy tissues or if L1 mobilization is restricted to neuronal precursor cells (NPCs) in the human brain. Here, we took advantage of an engineered L1 retrotransposition assay to analyze L1 mobilization rates in human mesenchymal (MSCs) and hematopoietic (HSCs) somatic stem cells. Notably, we have observed that L1 expression and engineered retrotransposition is much lower in both MSCs and HSCs when compared to NPCs. Remarkably, we have further demonstrated for the first time that engineered L1s can retrotranspose efficiently in mature nondividing neuronal cells. Thus, these findings suggest that the degree of somatic mosaicism and the impact of L1 retrotransposition in the human brain is likely much higher than previously thought. PMID:27965292

Somatic embryogenesis and plant regeneration from cell suspension cultures of Cucumis sativus L.

PubMed

Chee, P P; Tricoli, D M

1988-06-01

A procedure for the regeneration of whole cucumber plants (Cucumis sativus L. cv. Poinsett 76) by embryogenesis from cell suspension cultures is described. Embryogenic callus was initiated from the primary leaves of 14-17 day old plants. Suspension cultures of embryogenic cells were grown in liquid Murashige and Skoog basal medium containing 5 uM 2,4,5-trichlorophenoxyacetic acid and 4 uM 6-benzylaminopurine. Suspension cultures were composed of a population of cells that were densely cytoplasmic and potentially embryogenic. Differentiation of embryos was enhanced by washing the suspension culture cells with MS basal medium containing 0.5% activated charcoal and twice with MS basal medium followed by liquid shake cultures in MS basal medium. Sixty to 70 percent of the embryos prewashed with activated charcoal germinated into plantlets with normal morphology. Embryos obtained from suspension cultured cells without prewashing with activated charcoal organized into plantlets with abnormal primary leaves. Morphologically normal plantlets were obtained by excising the shoot tips and transferring them to fresh medium.

The role of silicon in plant tissue culture

PubMed Central

Sivanesan, Iyyakkannu; Park, Se Won

2014-01-01

Growth and morphogenesis of in vitro cultures of plant cells, tissues, and organs are greatly influenced by the composition of the culture medium. Mineral nutrients are necessary for the growth and development of plants. Several morpho-physiological disorders such as hooked leaves, hyperhydricity, fasciation, and shoot tip necrosis are often associated with the concentration of inorganic nutrient in the tissue culture medium. Silicon (Si) is the most abundant mineral element in the soil. The application of Si has been demonstrated to be beneficial for growth, development and yield of various plants and to alleviate various stresses including nutrient imbalance. Addition of Si to the tissue culture medium improves organogenesis, embryogenesis, growth traits, morphological, anatomical, and physiological characteristics of leaves, enhances tolerance to low temperature and salinity, protects cells and against metal toxicity, prevents oxidative phenolic browning and reduces the incidence of hyperhydricity in various plants. Therefore, Si possesses considerable potential for application in a wide range of plant tissue culture studies such as cryopreservation, organogenesis, micropropagation, somatic embryogenesis and secondary metabolites production. PMID:25374578

Linking maternal and somatic 5S rRNA types with different sequence-specific non-LTR retrotransposons

PubMed Central

Pagano, Johanna F.B.; Ensink, Wim A.; van Olst, Marina; van Leeuwen, Selina; Nehrdich, Ulrike; Zhu, Kongju; Spaink, Herman P.; Girard, Geneviève; Rauwerda, Han; Jonker, Martijs J.; Dekker, Rob J.

2017-01-01

5S rRNA is a ribosomal core component, transcribed from many gene copies organized in genomic repeats. Some eukaryotic species have two 5S rRNA types defined by their predominant expression in oogenesis or adult tissue. Our next-generation sequencing study on zebrafish egg, embryo, and adult tissue identified maternal-type 5S rRNA that is exclusively accumulated during oogenesis, replaced throughout the embryogenesis by a somatic-type, and thus virtually absent in adult somatic tissue. The maternal-type 5S rDNA contains several thousands of gene copies on chromosome 4 in tandem repeats with small intergenic regions, whereas the somatic-type is present in only 12 gene copies on chromosome 18 with large intergenic regions. The nine-nucleotide variation between the two 5S rRNA types likely affects TFIII binding and riboprotein L5 binding, probably leading to storage of maternal-type rRNA. Remarkably, these sequence differences are located exactly at the sequence-specific target site for genome integration by the 5S rRNA-specific Mutsu retrotransposon family. Thus, we could define maternal- and somatic-type MutsuDr subfamilies. Furthermore, we identified four additional maternal-type and two new somatic-type MutsuDr subfamilies, each with their own target sequence. This target-site specificity, frequently intact maternal-type retrotransposon elements, plus specific presence of Mutsu retrotransposon RNA and piRNA in egg and adult tissue, suggest an involvement of retrotransposons in achieving the differential copy number of the two types of 5S rDNA loci. PMID:28003516

Do early changes in the HAM-D-17 anxiety/somatization factor items affect treatment outcome among depressed outpatients? Comparison of two controlled trials of St John’s Wort (Hypericum Perforatum) versus an SSRI

PubMed Central

Bitran, Stella; Farabaugh, Amy H; Ameral, Victoria E; LaRocca, Rachel A; Clain, Alisabet J; Fava, Maurizio; Mischoulon, David

2011-01-01

Objective To assess whether early changes in HAM-D-17 anxiety/somatization items predict remission in two controlled studies of hypericum perforatum (St. John’s wort) versus an SSRI for major depressive disorder (MDD). Methods The Hypericum Depression Trial Study Group (NIMH) study randomized 340 subjects to hypericum, sertraline, or placebo for 8 weeks. The MGH study randomized 135 subjects to hypericum, fluoxetine, or placebo for 12 weeks. We examined whether remission was associated with early changes in anxiety/somatization symptoms. Results In the NIMH study, significant associations were observed between remission and early improvement in the anxiety-psychic item (sertraline arm), somatic-gastrointestinal item (hypericum arm), and somatic symptoms-general (placebo arm). None of the three treatment arms of the MGH study showed significant associations between anxiety/somatization symptoms and remission. When both study samples were pooled, we found associations for anxiety-psychic (SSRI arm), somatic-gastrointestinal and hypochondriasis (hypericum arm), and anxiety-psychic and somatic symptoms-general (placebo arm). In the entire sample, remission was associated with improvement in the anxiety-psychic, somatic-gastrointestinal, and somatic symptoms-general items. Conclusions The number and type of anxiety/somatization items associated with remission varied depending on the intervention. Early scrutiny of the HAM-D-17 anxiety/somatization items may help predict remission of MDD. PMID:21278577

Biolistics Transformation of Wheat

NASA Astrophysics Data System (ADS)

Sparks, Caroline A.; Jones, Huw D.

We present a complete, step-by-step guide to the production of transformed wheat plants using a particle bombardment device to deliver plasmid DNA into immature embryos and the regeneration of transgenic plants via somatic embryogenesis. Currently, this is the most commonly used method for transforming wheat and it offers some advantages. However, it will be interesting to see whether this position is challenged as facile methods are developed for delivering DNA by Agrobacterium tumefaciens or by the production of transformants via a germ-line process (see other chapters in this book).

Cloning crops in a CELSS via tissue culture: Prospects and problems

NASA Technical Reports Server (NTRS)

Carman, John G.; Hess, J. Richard

1990-01-01

Micropropagation is currently used to clone fruits, nuts, and vegetables and involves controlling the outgrowth in vitro of basal, axillary, or adventitious buds. Following clonal multiplication, shoots are divided and rooted. This process has greatly reduced space and energy requirements in greenhouses and field nurseries and has increased multiplication rates by greater than 20 fold for some vegetatively propagated crops and breeding lines. Cereal and legume crops can also be cloned by tissue culture through somatic embryogenesis. Somatic embryos can be used to produce 'synthetic seed', which can tolerate desiccation and germinate upon rehydration. Synthetic seed of hybrid wheat, rice, soybean and other crops could be produced in a controlled ecological life support system. Thus, yield advantages of hybreds over inbreds (10 to 20 percent) could be exploited without having to provide additional facilities and energy for parental-line and hybrid seed nurseries.

Morphohistobiochemical characteristics of embryogenic and nonembryogenic callus cultures of sweet potato (Ipomoea batatas L.).

PubMed

Mukherjee, A; Debata, B K; Mukherjee, P S; Malik, S K

2001-01-01

Ipomoea batatas callus culture raised in a medium supplemented with 2,4-D (2,4-dichlorophenoxy acetic acid) alone or 2,4-D in combination with benzyl adenine, were found to be embryogenic. Supplementation of exogenous chemicals, such as 5 g/l NaCI or 0.7 g/l proline together with a mild dose of 0.2 mg/l 2,4-D, enhanced somatic embryogenesis significantly in all the genotypes tested. Morphological, growth, physiological, histological, and biochemical characteristics of the embryogenic callus were different from the nonembryogenic callus. The former was compact, slow growing, and nodular compared with the fast growing, fragile, nonembryogenic callus. The embryogenic callus tissue had more dry matter, protein and reducing sugar contents compared with the less embryogenic callus. The somatic embryogenic response remained steady in the cultures for up to 96 weeks.

Dissection and staining of Drosophila larval ovaries.

PubMed

Maimon, Iris; Gilboa, Lilach

2011-05-13

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) (1, 2). The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches (3-12). Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar (13-17). GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs (7, 16, 18, 19). Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism. Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100μm across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Intranuclear DNA density affects chromosome condensation in metazoans

PubMed Central

Hara, Yuki; Iwabuchi, Mari; Ohsumi, Keita; Kimura, Akatsuki

2013-01-01

Chromosome condensation is critical for accurate inheritance of genetic information. The degree of condensation, which is reflected in the size of the condensed chromosomes during mitosis, is not constant. It is differentially regulated in embryonic and somatic cells. In addition to the developmentally programmed regulation of chromosome condensation, there may be adaptive regulation based on spatial parameters such as genomic length or cell size. We propose that chromosome condensation is affected by a spatial parameter called the chromosome amount per nuclear space, or “intranuclear DNA density.” Using Caenorhabditis elegans embryos, we show that condensed chromosome sizes vary during early embryogenesis. Of importance, changing DNA content to haploid or polyploid changes the condensed chromosome size, even at the same developmental stage. Condensed chromosome size correlates with interphase nuclear size. Finally, a reduction in nuclear size in a cell-free system from Xenopus laevis eggs resulted in reduced condensed chromosome sizes. These data support the hypothesis that intranuclear DNA density regulates chromosome condensation. This suggests an adaptive mode of chromosome condensation regulation in metazoans. PMID:23783035

Ribosomal stress induces L11- and p53-dependent apoptosis in mouse pluripotent stem cells.

PubMed

Morgado-Palacin, Lucia; Llanos, Susana; Serrano, Manuel

2012-02-01

Ribosome biogenesis is the most demanding energetic process in proliferating cells and it is emerging as a critical sensor of cellular homeostasis. Upon disturbance of ribosome biogenesis, specific free ribosomal proteins, most notably L11, bind and inhibit Mdm2, resulting in activation of the tumor suppressor p53. This pathway has been characterized in somatic and cancer cells, but its function in embryonic pluripotent cells has remained unexplored. Here, we show that treatment with low doses of Actinomycin D or depletion of ribosomal protein L37, two well-established inducers of ribosomal stress, activate p53 in an L11-dependent manner in mouse embryonic stem cells (ESCs) and in induced pluripotent stem cells (iPSCs). Activation of p53 results in transcriptional induction of p53 targets, including p21, Mdm2, Pidd, Puma, Noxa and Bax. Finally, ribosomal stress elicits L11- and p53-dependent apoptosis in ESCs/iPSCs. These results extend to pluripotent cells the functionality of the ribosomal stress pathway and we speculate that this could be a relevant cellular checkpoint during early embryogenesis.

Do early changes in the HAM-D-17 anxiety/somatization factor items affect the treatment outcome among depressed outpatients? Comparison of two controlled trials of St John's wort (Hypericum perforatum) versus a SSRI.

PubMed

Bitran, Stella; Farabaugh, Amy H; Ameral, Victoria E; LaRocca, Rachel A; Clain, Alisabet J; Fava, Maurizio; Mischoulon, David

2011-07-01

To assess whether early changes in Hamilton Depression Rating Scale-17 anxiety/somatization items predict remission in two controlled studies of Hypericum perforatum (St John's wort) versus selective serotonin reuptake inhibitors for major depressive disorder. The Hypericum Depression Trial Study Group (National Institute of Mental Health) randomized 340 patients to Hypericum, sertraline, or placebo for 8 weeks, whereas the Massachusetts General Hospital study randomized 135 patients to Hypericum, fluoxetine, or placebo for 12 weeks. The investigators examined whether remission was associated with early changes in anxiety/somatization symptoms. In the National Institute of Mental Health study, significant associations were observed between remission and early improvement in the anxiety (psychic) item (sertraline arm), somatic (gastrointestinal item; Hypericum arm), and somatic (general) symptoms (placebo arm). None of the three treatment arms of the Massachusetts General Hospital study showed significant associations between anxiety/somatization symptoms and remission. When both study samples were pooled, we found associations for anxiety (psychic; selective serotonin reuptake inhibitors arm), somatic (gastrointestinal), and hypochondriasis (Hypericum arm), and anxiety (psychic) and somatic (general) symptoms (placebo arm). In the entire sample, remission was associated with the improvement in the anxiety (psychic), somatic (gastrointestinal), and somatic (general) items. The number and the type of anxiety/somatization items associated with remission varied depending on the intervention. Early scrutiny of the Hamilton Depression Rating Scale-17 anxiety/somatization items may help to predict remission of major depressive disorder.

Fibroblast growth factor signaling is required for early somatic gonad development in zebrafish.

PubMed

Leerberg, Dena M; Sano, Kaori; Draper, Bruce W

2017-09-01

The vertebrate ovary and testis develop from a sexually indifferent gonad. During early development of the organism, primordial germ cells (the gamete lineage) and somatic gonad cells coalesce and begin to undergo growth and morphogenesis to form this bipotential gonad. Although this aspect of development is requisite for a fertile adult, little is known about the genetic regulation of early gonadogenesis in any vertebrate. Here, we provide evidence that fibroblast growth factor (Fgf) signaling is required for the early growth phase of a vertebrate bipotential gonad. Based on mutational analysis in zebrafish, we show that the Fgf ligand 24 (Fgf24) is required for proliferation, differentiation, and morphogenesis of the early somatic gonad, and as a result, most fgf24 mutants are sterile as adults. Additionally, we describe the ultrastructural elements of the early zebrafish gonad and show that distinct somatic cell populations can be identified soon after the gonad forms. Specifically, we show that fgf24 is expressed in an epithelial population of early somatic gonad cells that surrounds an inner population of mesenchymal somatic gonad cells that are in direct contact with the germ cells, and that fgf24 is required for stratification of the somatic tissue. Furthermore, based on gene expression analysis, we find that differentiation of the inner mesenchymal somatic gonad cells into functional cell types in the larval and early juvenile-stage gonad is dependent on Fgf24 signaling. Finally, we argue that the role of Fgf24 in zebrafish is functionally analogous to the role of tetrapod FGF9 in early gonad development.

Effect of genotype, gelling agent, and auxin on the induction of somatic embryogenesis in sweet potato (Ipomoea batatas Lam.).

PubMed

El Abidine Triqui, Zine; Guédira, Abdelkarim; Chlyah, Averil; Chlyah, Hassane; Souvannavong, Vongthip; Haïcour, Robert; Sihachakr, Darasinh

2008-03-01

Lateral buds of six cultivars of sweet potato were induced to form embryogenic callus in a culture medium solidified with two types of gelling agents, Agar or Gelrite, and supplemented with various concentrations of auxins, 2,4-D, 2,4,5-T and Picloram. Of the six cultivars screened, only three gave an embryogenic response. Best results with an average of 3.53% embryogenic response were obtained with the medium solidified with Agar, while in Gelrite only 0.45% of lateral buds gave rise to embryogenic callus. The interaction between the genotype and auxins was highly significant; particularly the optimal response was obtained with cv. Zho and 865 yielding 10.7 and 14.7% somatic embryogenesis, respectively, in the medium containing 2,4,5-T or Picloram. The plant conversion was dramatically improved by subculture of the embryogenic callus on the medium with the combination of 1 microM 2,4-D and 1 microM Kinetin or 5 microM ABA alone before transfer of mature embryos onto hormone-free medium. The embryogenic callus of sweet potato and its sustained ability to further regenerate plants have regularly been maintained for several years by frequent subculture in 5 microM 2,4,5-T or the combination of 10 microM 2,4-D and 1 microM BAP or kinetin. The embryo-derived plants seemed apparently genetically stable and similar to the hexaploid parental plants, based on morphological analysis and their ploidy level determined by using flow cytometry.

Characterization of the third SERK gene in pineapple (Ananas comosus) and analysis of its expression and autophosphorylation activity in vitro

PubMed Central

Ma, Jun; He, Yehua; Hu, Zhongyi; Xu, Wentian; Xia, Jingxian; Guo, Cuihong; Lin, Shunquan; Chen, Chengjie; Wu, Chenghou; Zhang, Junli

2014-01-01

Two somatic embryogenesis receptor-like kinase genes (identified as AcSERK1 and AcSERK2) have previously been characterized from pineapple (Ananas comosus). In this work, we describe the characterization of a third gene (AcSERK3) in this family. AcSERK3 had all the characteristic domains and shared extensive sequence homology with other plant SERKs. AcSERK3 expression was studied by in situ hybridization and quantitative real-time PCR to analyze its function. Intense in situ hybridization signals were observed only in single competent cells and competent cell clusters; no hybridization signal was detected in the subsequent stages of somatic embryogenesis. AcSERK3 was highly expressed in embryogenic callus compared to other organs, e.g., 20–80 fold more than in anther but similar to that of non-embryogenic callus, which was 20–50 fold that of anther. AcSERK3 expression in root was 80 fold higher than in anther and the highest amongst all organs tested. These results indicate that AcSERK3 plays an important role in callus proliferation and root development. His-tagged AcSERK3 protein was successfully expressed and the luminescence of His6-AcSERK3 protein was only ∼5% of that of inactivated AcSERK3 protein and reaction buffer without protein, and 11.3% of that of an extract of host Escherichia coli pET-30a. This finding confirmed that the AcSERK3 fusion protein had autophosphorylation activity. PMID:25249776

Linking maternal and somatic 5S rRNA types with different sequence-specific non-LTR retrotransposons.

PubMed

Locati, Mauro D; Pagano, Johanna F B; Ensink, Wim A; van Olst, Marina; van Leeuwen, Selina; Nehrdich, Ulrike; Zhu, Kongju; Spaink, Herman P; Girard, Geneviève; Rauwerda, Han; Jonker, Martijs J; Dekker, Rob J; Breit, Timo M

2017-04-01

5S rRNA is a ribosomal core component, transcribed from many gene copies organized in genomic repeats. Some eukaryotic species have two 5S rRNA types defined by their predominant expression in oogenesis or adult tissue. Our next-generation sequencing study on zebrafish egg, embryo, and adult tissue identified maternal-type 5S rRNA that is exclusively accumulated during oogenesis, replaced throughout the embryogenesis by a somatic-type, and thus virtually absent in adult somatic tissue. The maternal-type 5S rDNA contains several thousands of gene copies on chromosome 4 in tandem repeats with small intergenic regions, whereas the somatic-type is present in only 12 gene copies on chromosome 18 with large intergenic regions. The nine-nucleotide variation between the two 5S rRNA types likely affects TFIII binding and riboprotein L5 binding, probably leading to storage of maternal-type rRNA. Remarkably, these sequence differences are located exactly at the sequence-specific target site for genome integration by the 5S rRNA-specific Mutsu retrotransposon family. Thus, we could define maternal- and somatic-type MutsuDr subfamilies. Furthermore, we identified four additional maternal-type and two new somatic-type MutsuDr subfamilies, each with their own target sequence. This target-site specificity, frequently intact maternal-type retrotransposon elements, plus specific presence of Mutsu retrotransposon RNA and piRNA in egg and adult tissue, suggest an involvement of retrotransposons in achieving the differential copy number of the two types of 5S rDNA loci. © 2017 Locati et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Engineered LINE-1 retrotransposition in nondividing human neurons.

PubMed

Macia, Angela; Widmann, Thomas J; Heras, Sara R; Ayllon, Veronica; Sanchez, Laura; Benkaddour-Boumzaouad, Meriem; Muñoz-Lopez, Martin; Rubio, Alejandro; Amador-Cubero, Suyapa; Blanco-Jimenez, Eva; Garcia-Castro, Javier; Menendez, Pablo; Ng, Philip; Muotri, Alysson R; Goodier, John L; Garcia-Perez, Jose L

2017-03-01

Half the human genome is made of transposable elements (TEs), whose ongoing activity continues to impact our genome. LINE-1 (or L1) is an autonomous non-LTR retrotransposon in the human genome, comprising 17% of its genomic mass and containing an average of 80-100 active L1s per average genome that provide a source of inter-individual variation. New LINE-1 insertions are thought to accumulate mostly during human embryogenesis. Surprisingly, the activity of L1s can further impact the somatic human brain genome. However, it is currently unknown whether L1 can retrotranspose in other somatic healthy tissues or if L1 mobilization is restricted to neuronal precursor cells (NPCs) in the human brain. Here, we took advantage of an engineered L1 retrotransposition assay to analyze L1 mobilization rates in human mesenchymal (MSCs) and hematopoietic (HSCs) somatic stem cells. Notably, we have observed that L1 expression and engineered retrotransposition is much lower in both MSCs and HSCs when compared to NPCs. Remarkably, we have further demonstrated for the first time that engineered L1s can retrotranspose efficiently in mature nondividing neuronal cells. Thus, these findings suggest that the degree of somatic mosaicism and the impact of L1 retrotransposition in the human brain is likely much higher than previously thought. © 2017 Macia et al.; Published by Cold Spring Harbor Laboratory Press.

RELATIONSHIPS BETWEEN BREAST-FEEDING, CO-SLEEPING, AND SOMATIC COMPLAINTS IN EARLY CHILDHOOD.

PubMed

Peters, Elisabeth Maria; Lusher, Joanne Marie; Banbury, Samantha; Chandler, Chris

2016-09-01

The central aim of this study was to expand a limited body of knowledge on the complex relationship between breast-feeding, co-sleeping, and somatic complaints in early childhood. An opportunity sample of 98 parents from the general population with children aged 18 to 60 months consented to participate in the study. Each parent completed a series of questionnaires measuring somatic complaints, sleep problems, co-sleeping, breast-feeding, and demographic factors. Findings indicated that co-sleeping was associated with increased somatic complaints and that breast-feeding associated with decreased somatic complaints. Co-sleeping also was found to be associated with an increase in sleep problems. Boys demonstrated significantly higher levels of sleep problems than did girls. These findings highlight the relationship between co-sleeping during early childhood, which could have implications for prevention, treatment, and intervention regarding somatic complaints and sleep problems in early childhood. © 2016 Michigan Association for Infant Mental Health.

Ultra-deep sequencing of ribosome-associated poly-adenylated RNA in early Drosophila embryos reveals hundreds of conserved translated sORFs.

PubMed

Li, Hongmei; Hu, Chuansheng; Bai, Ling; Li, Hua; Li, Mingfa; Zhao, Xiaodong; Czajkowsky, Daniel M; Shao, Zhifeng

2016-12-01

There is growing recognition that small open reading frames (sORFs) encoding peptides shorter than 100 amino acids are an important class of functional elements in the eukaryotic genome, with several already identified to play critical roles in growth, development, and disease. However, our understanding of their biological importance has been hindered owing to the significant technical challenges limiting their annotation. Here we combined ultra-deep sequencing of ribosome-associated poly-adenylated RNAs with rigorous conservation analysis to identify a comprehensive population of translated sORFs during early Drosophila embryogenesis. In total, we identify 399 sORFs, including those previously annotated but without evidence of translational capacity, those found within transcripts previously classified as non-coding, and those not previously known to be transcribed. Further, we find, for the first time, evidence for translation of many sORFs with different isoforms, suggesting their regulation is as complex as longer ORFs. Furthermore, many sORFs are found not associated with ribosomes in late-stage Drosophila S2 cells, suggesting that many of the translated sORFs may have stage-specific functions during embryogenesis. These results thus provide the first comprehensive annotation of the sORFs present during early Drosophila embryogenesis, a necessary basis for a detailed delineation of their function in embryogenesis and other biological processes. © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Somatic mosaicism in a case of apparently sporadic Creutzfeldt-Jakob disease carrying a de novo D178N mutation in the PRNP gene.

PubMed

Alzualde, A; Moreno, F; Martínez-Lage, P; Ferrer, I; Gorostidi, A; Otaegui, D; Blázquez, L; Atares, B; Cardoso, S; Martínez de Pancorbo, M; Juste, R; Rodríguez-Martínez, A B; Indakoetxea, B; López de Munain, A

2010-10-05

Transmissible spongiform encephalopathies (TSEs) are a group of rare fatal neurodegenerative disorders. Creutzfeldt-Jakob disease (CJD) represents the most common form of TSE and can be classified into sporadic, genetic, iatrogenic and variant forms. Genetic cases are related to prion protein gene mutations but they only account for 10-20% of cases. Here we report an apparently sporadic CJD case with negative family history carrying a mutation at codon 178 of prion protein gene. This mutation is a de novo mutation as the parents of the case do not show it. Furthermore the presence of three different alleles (wild type 129M-178D and 129V-178D and mutated 129V-178N), confirmed by different methods, indicates that this de novo mutation is a post-zygotic mutation that produces somatic mosaicism. The proportion of mutated cells in peripheral blood cells and in brain tissue was similar and was estimated at approximately 97%, suggesting that the mutation occurred at an early stage of embryogenesis. Neuropathological examination disclosed spongiform change mainly involving the caudate and putamen, and the cerebral cortex, together with proteinase K-resistant PrP globular deposits in the cerebrum and cerebellum. PrP typing was characterized by a lower band of 21 kDa. This is the first case of mosaicism described in prion diseases and illustrates a potential etiology for apparently sporadic neurodegenerative diseases. In light of this case, genetic counseling for inherited and sporadic forms of transmissible encephalopathies should take into account this possibility for genetic screening procedures.

Xanthan gum: an economical substitute for agar in plant tissue culture media.

PubMed

Jain, R; Babbar, S B

2006-03-01

Xanthan gum, a microbial desiccation-resistant polysaccharide prepared commercially by aerobic submerged fermentation from Xanthomonas campestris, has been successfully used as a solidifying agent for plant tissue culture media. Its suitability as a substitute to agar was demonstrated for in vitro seed germination, caulogenesis and rhizogenesis of Albizzia lebbeck, androgenesis in anther cultures of Datura innoxia, and somatic embryogenesis in callus cultures of Calliandra tweedii. Culture media used for eliciting these morphogenic responses were gelled with either 1% xanthan gum or 0.9% agar. Xanthan gum, like agar, supported all these responses.

Somatic embryogenesis and plant regeneration in tissue cultures of sweet potato (Ipomea batatas Poir.).

PubMed

Liu, J R; Cantliffe, D J

1984-06-01

Leaf, shoot-tip, stem, and root explants of sweet potato (Ipomea batatas Poir.) gave rise to two kinds of callus on nutrient agar medium containing 0.5 to 2.0 mg/l 2,4-D. One callus, bright- to pale-yellow, was compact and organized, while the other was dull-yellow and friable. The former callus gave rise to numerous globular and heart-shaped embryoids. When transferred onto hormone-free medium, the embryoids readily developed into a torpedo-shape before germination. The plantlets were transplanted to soil where they flowered and formed storage roots at maturity.

Expression of the adhesion G protein-coupled receptor A2 (adgra2) during Xenopus laevis development.

PubMed

Seigfried, Franziska A; Dietmann, Petra; Kühl, Michael; Kühl, Susanne J

2018-06-01

The adhesion G protein-coupled receptor A2 (Adgra2) is a seven transmembrane receptor that has been described to be a regulator for angiogenesis in mice. Furthermore, the zebrafish ouchless mutant is unable to develop dorsal root ganglia through a disrupted trafficking of Adgra2. Besides RNA sequencing data, nothing is reported about Adgra2 in the south African crawled frog Xenopus laevis. In this study, we investigated for the first time the spatio-temporal expression of adgra2 during early Xenopus embryogenesis in detail. In silico approaches showed that the genomic adgra2 region as well as the Adgra2 protein sequence is highly conserved among different species including Xenopus. RT-PCR experiments confirmed that embryonic adgra2 expression is primarily detected at the beginning of neurulation and is then present throughout the whole Xenopus embryogenesis until stage 42. Whole mount in situ hybridization approaches visualized adgra2 expression in many tissues during Xenopus embryogenesis such as the cardiovascular system including the heart, the migrating neural crest cells and the developing eye including the periocular mesenchyme. Our results indicate a role of Adgra2 for embryogenesis and are a good starting point for further functional studies during early vertebrate development. Copyright © 2018 Elsevier B.V. All rights reserved.

Zebrafish E-cadherin: expression during early embryogenesis and regulation during brain development.

PubMed

Babb, S G; Barnett, J; Doedens, A L; Cobb, N; Liu, Q; Sorkin, B C; Yelick, P C; Raymond, P A; Marrs, J A

2001-06-01

Zebrafish E-cadherin (cdh1) cell adhesion molecule cDNAs were cloned. We investigated spatial and temporal expression of cdh1 during early embryogenesis. Expression was observed in blastomeres, the anterior mesoderm during gastrulation, and developing epithelial structures. In the developing nervous system, cdh1 was detected at the pharyngula stage (24 hpf) in the midbrain-hindbrain boundary (MHB). Developmental regulation of MHB formation involves wnt1 and pax2.1. wnt1 expression preceded cdh1 expression during MHB formation, and cdh1 expression in the MHB was dependent on normal development of this structure. Copyright 2001 Wiley-Liss, Inc.

Convergent occurrence of the developmental hourglass in plant and animal embryogenesis?

PubMed

Cridge, Andrew G; Dearden, Peter K; Brownfield, Lynette R

2016-04-01

The remarkable similarity of animal embryos at particular stages of development led to the proposal of a developmental hourglass. In this model, early events in development are less conserved across species but lead to a highly conserved 'phylotypic period'. Beyond this stage, the model suggests that development once again becomes less conserved, leading to the diversity of forms. Recent comparative studies of gene expression in animal groups have provided strong support for the hourglass model. How and why might such an hourglass pattern be generated? More importantly, how might early acting events in development evolve while still maintaining a later conserved stage? The discovery that an hourglass pattern may also exist in the embryogenesis of plants provides comparative data that may help us explain this phenomenon. Whether the developmental hourglass occurs in plants, and what this means for our understanding of embryogenesis in plants and animals is discussed. Models by which conserved early-acting genes might change their functional role in the evolution of gene networks, how networks buffer these changes, and how that might constrain, or confer diversity, of the body plan are also discused. Evidence of a morphological and molecular hourglass in plant and animal embryogenesis suggests convergent evolution. This convergence is likely due to developmental constraints imposed upon embryogenesis by the need to produce a viable embryo with an established body plan, controlled by the architecture of the underlying gene regulatory networks. As the body plan is largely laid down during the middle phases of embryo development in plants and animals, then it is perhaps not surprising this stage represents the narrow waist of the hourglass where the gene regulatory networks are the oldest and most robust and integrated, limiting species diversity and constraining morphological space. © The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company.

Glycogen and Glucose Metabolism Are Essential for Early Embryonic Development of the Red Flour Beetle Tribolium castaneum

PubMed Central

Fraga, Amanda; Ribeiro, Lupis; Lobato, Mariana; Santos, Vitória; Silva, José Roberto; Gomes, Helga; da Cunha Moraes, Jorge Luiz; de Souza Menezes, Jackson

2013-01-01

Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis) and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3) and hexokinase (HexA) genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen. PMID:23750237

Regulation of early Xenopus development by ErbB signaling

PubMed Central

Nie, Shuyi; Chang, Chenbei

2008-01-01

ErbB signaling has long been implicated in cancer formation and progression and is shown to regulate cell division, migration and death during tumorigenesis. The functions of the ErbB pathway during early vertebrate embryogenesis, however, are not well understood. Here we report characterization of ErbB activities during early frog development. Gain-of-function analyses show that EGFR, ErbB2 and ErbB4 induce ectopic tumor-like cell mass that contains increased numbers of mitotic cells. Both the muscle and the neural markers are expressed in these ectopic protrusions. ErbBs also induce mesodermal markers in ectodermal explants. Loss-of-function studies using carboxyl terminal-truncated dominant-negative ErbB receptors demonstrate that blocking ErbB signals leads to defective gastrulation movements and malformation of the embryonic axis with a reduction in the head structures in early frog embryos. These data, together with the observation that ErbBs are expressed early during frog embryogenesis, suggest that ErbBs regulate cell proliferation, movements and embryonic patterning during early Xenopus development. PMID:16258939

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

The role of the transformer gene in sex determination and reproduction in the tephritid fruit fly, Bactrocera dorsalis (Hendel).

PubMed

Peng, Wei; Zheng, Wenping; Handler, Alfred M; Zhang, Hongyu

2015-12-01

Transformer (tra) is a switch gene in the somatic sex-determination hierarchy that regulates sexual dimorphism based on RNA splicing in many insects. In tephritids, a Y-linked male determining gene (M) controls sex in the sex-determination pathway. Here, homologues of Drosophila tra and transformer-2 (tra-2) genes were isolated and characterized in Bactrocera dorsalis (Hendel), one of the most destructive agricultural insect pests in many Asian countries. Two male-specific and one female-specific isoforms of B. dorsalis transformer (Bdtra) were identified. The presence of multiple TRA/TRA-2 binding sites in Bdtra suggests that the TRA/TRA-2 proteins are splicing regulators promoting and maintaining, epigenetically, female sex determination by a tra positive feedback loop in XX individuals during development. The expression patterns of female-specific Bdtra transcripts during early embryogenesis shows that a peak appears at 15 h after egg laying. Using dsRNA to knock-down Bdtra expression in the embryo and adult stages, we showed that sexual formation is determined early in the embryo stage and that parental RNAi does not lead to the production of all male progeny as in Tribolium castaneum. RNAi results from adult abdominal dsRNA injections show that Bdtra has a positive influence on female yolk protein gene (Bdyp1) expression and fecundity.

ADP-ribosyl cyclases regulate early development of the sea urchin.

PubMed

Ramakrishnan, Latha; Uhlinger, Kevin; Dale, Leslie; Hamdoun, Amro; Patel, Sandip

2016-06-01

ADP-ribosyl cyclases are multifunctional enzymes involved in the metabolism of nucleotide derivatives necessary for Ca 2+ signalling such as cADPR and NAADP. Although Ca 2+ signalling is a critical regulator of early development, little is known of the role of ADP-ribosyl cyclases during embryogenesis. Here we analyze the expression, activity and function of ADP-ribosyl cyclases in the embryo of the sea urchin - a key organism for study of both Ca 2+ signalling and embryonic development. ADP-ribosyl cyclase isoforms (SpARC1-4) showed unique changes in expression during early development. These changes were associated with an increase in the ratio of cADPR:NAADP production. Over-expression of SpARC4 (a preferential cyclase) disrupted gastrulation. Our data highlight the importance of ADP-ribosyl cyclases during embryogenesis.

The relationship between early changes in the HAMD-17 anxiety/somatization factor items and treatment outcome among depressed outpatients.

PubMed

Farabaugh, Amy; Mischoulon, David; Fava, Maurizio; Wu, Shirley L; Mascarini, Alessandra; Tossani, Eliana; Alpert, Jonathan E

2005-03-01

The 17-item Hamilton Rating Scale for Depression (HAMD-17) Anxiety/Somatization factor includes six items: Anxiety (psychic), Anxiety (somatic), Somatic Symptoms (gastrointestinal), Somatic Symptoms (general), Hypochondriasis and Insight. This study examines the relationship between early changes (defined as those observed between baseline and week 1) in these HAMD-17 Anxiety/Somatization Factor items and treatment outcome among major depressive disorder (MDD) patients who participated in a study comparing the antidepressant efficacy of a standardized extract of hypericum with both placebo and fluoxetine. Following a 1-week, single-blind washout, patients with MDD diagnosed by the Structured Clinical Interview for DSM-IV (SCID) were randomized to 12 weeks of double-blind treatment with hypericum extract (900 mg/day), fluoxetine (20 mg/day) or placebo. The relationship between early changes in HAMD-17 anxiety/somatization factor items and treatment outcome was assessed separately for patients who received study treatment (hypericum or fluoxetine) versus placebo with a logistic regression method. One hundred and thirty-five patients (female 57%, mean age=37.3+/-11.0 years; mean baseline HAMD-17=19.7+/-3.2 years) were randomized to double-blind treatment and were included in the intent-to-treat (ITT) analyses. After adjusting for baseline HAMD-17 scores and for multiple comparisons with the Bonferroni correction, patients who remitted (HAMD-17 score <8) after study treatment had significantly greater early improvement in Somatic Symptoms (General) scores than non-remitters. No other significant differences in early changes were noted for the remaining items between remitters versus non-remitters who received active treatment. For patients treated with placebo, early change was not predictive of remission for any of the items after Bonferroni correction. In conclusion, the presence of early improvement on the HAMD-17 item concerning fatigue and general somatic symptoms is significantly predictive of achieving remission at endpoint with active study treatment but not with placebo.

Anxious Depression and early changes in the HAMD-17 anxiety-somatization factor items and antidepressant treatment outcome

PubMed Central

Farabaugh, Amy H.; Bitran, Stella; Witte, Janet; Alpert, Jonathan; Chuzi, Sarah; Clain, Alisabet J.; Baer, Lee; Fava, Maurizio; McGrath, Patrick J.; Dording, Christina; Mischoulon, David; Papakostas, George I

2010-01-01

Objective To assess the relationship between early changes in anxiety/somatization symptoms and treatment outcome among MDD subjects during a 12-week trial of fluoxetine. We also examined the relationship between anxious depression and treatment response. Methods 510 MDD patients received 12 weeks of fluoxetine with flexible dosing (target dosages: 10 mg/day (week 1), 20 mg/day (weeks 2–4), 40 mg/day (weeks 4–8), and 60 mg/day (weeks 5–12)). We assessed the relationship between early changes in HAMD-17- anxiety/somatization factor items and depressive remission, as well as whether anxious depression at baseline predicted remission at study endpoint. . Baseline HAMD-17 scores were considered as covariates and the Bonferroni correction (p ≤ .008) was used for multiple comparisons. Results Adjusting for baseline HAMD-17 scores, patients who experienced greater early improvement in somatic symptoms (gastrointestinal) were significantly more likely to attain remission (HAMD-17 < 8) at endpoint than those without early improvement (p = .006). Early changes in the remaining items did not predict remission, nor did anxious depression at baseline. Conclusions Among the anxiety/somatization factor items, only early changes in somatic symptoms (gastrointestinal) predicted remission. Future studies are warranted to further investigate this relationship, as well as that between anxious depression and treatment outcome. PMID:20400905

Immunocytochemical localisation of the nucleolar protein fibrillarin and RNA polymerase I during mouse early embryogenesis.

PubMed

Cuadros-Fernández, J M; Esponda, P

1996-02-01

We have employed immunocytochemical procedures to localise the nucleolar protein fibrillarin and the enzyme RNA polymerase I in the numerous dense fibrillar bodies (nucleolar precursor bodies) which appear in the nuclei of mammalian early embryos. The aim of this study was to search for relationships between the localisation of these proteins, the changes in the structure of the nucleolar precursor bodies and the resumption of rRNA gene transcription during mouse early embryogenesis. Three human autoimmune sera which recognised fibrillarin and a rabbit antiserum created against RNA polymerase I were employed for fluorescence and electron microscopic immunocytochemical assays. A statistical analysis was also applied. Immunocytochemistry revealed that fibrillarin and RNA polymerase I showed the same localisation in the nucleolar precursor bodies. These proteins were immunolocalised only from the late 2-cell stage onward. Fibrillarin was initially detected at the periphery of the nucleolar precursor bodies and the labelling gradually increased until the morula and blastocyst stages, where normally active nucleoli are found. The pattern of increase of fibrillarin during early embryogenesis shows a parallelism with the rise in rRNA gene transcription occurring during these embryonic stages, and a possible correlation between these two phenomena is suggested. Results demonstrated that nucleolar precursor bodies differ in their biochemical composition from the nucleolus and also from the prenucleolar bodies which appear during mitosis. When anti-fibrillarin antibodies were microinjected into the male pronucleus of mouse embryos to analyse the functions of fibrillarin during early development, they partially blocked the early development of mouse embryos and only 23.8% of injected embryos reach the blastocyst stage.

Germline-specific H1 variants: the "sexy" linker histones.

PubMed

Pérez-Montero, Salvador; Carbonell, Albert; Azorín, Fernando

2016-03-01

The eukaryotic genome is packed into chromatin, a nucleoprotein complex mainly formed by the interaction of DNA with the abundant basic histone proteins. The fundamental structural and functional subunit of chromatin is the nucleosome core particle, which is composed by 146 bp of DNA wrapped around an octameric protein complex formed by two copies of each core histone H2A, H2B, H3, and H4. In addition, although not an intrinsic component of the nucleosome core particle, linker histone H1 directly interacts with it in a monomeric form. Histone H1 binds nucleosomes near the exit/entry sites of linker DNA, determines nucleosome repeat length and stabilizes higher-order organization of nucleosomes into the ∼30 nm chromatin fiber. In comparison to core histones, histone H1 is less well conserved through evolution. Furthermore, histone H1 composition in metazoans is generally complex with most species containing multiple variants that play redundant as well as specific functions. In this regard, a characteristic feature is the presence of specific H1 variants that replace somatic H1s in the germline and during early embryogenesis. In this review, we summarize our current knowledge about their structural and functional properties.

Drosophila Lin-52 Acts in Opposition to Repressive Components of the Myb-MuvB/dREAM Complex

PubMed Central

Lewis, Peter W.; Sahoo, Debashis; Geng, Cuiyun; Bell, Maren

2012-01-01

The Drosophila melanogaster Myb-MuvB/dREAM complex (MMB/dREAM) participates in both the activation and repression of developmentally regulated genes and origins of DNA replication. Mutants in MMB subunits exhibit diverse phenotypes, including lethality, eye defects, reduced fecundity, and sterility. Here, we used P-element excision to generate mutations in lin-52, which encodes the smallest subunit of the MMB/dREAM complex. lin-52 is required for viability, as null mutants die prior to pupariation. The generation of somatic and germ line mutant clones indicates that lin-52 is required for adult eye development and for early embryogenesis via maternal effects. Interestingly, the maternal-effect embryonic lethality, larval lethality, and adult eye defects could be suppressed by mutations in other subunits of the MMB/dREAM complex. These results suggest that a partial MMB/dREAM complex is responsible for the lethality and eye defects of lin-52 mutants. Furthermore, these findings support a model in which the Lin-52 and Myb proteins counteract the repressive activities of the other members of the MMB/dREAM complex at specific genomic loci in a developmentally controlled manner. PMID:22688510

Molecular characterization of cDNAs encoding G protein alpha and beta subunits and study of their temporal and spatial expression patterns in Nicotiana plumbaginifolia Viv.

PubMed

Kaydamov, C; Tewes, A; Adler, K; Manteuffel, R

2000-04-25

We have isolated cDNA sequences encoding alpha and beta subunits of potential G proteins from a cDNA library prepared from somatic embryos of Nicotiana plumbaginifolia Viv. at early developmental stages. The predicted NPGPA1 and NPGPB1 gene products are 75-98% identical to the known respective plant alpha and beta subunits. Southern hybridizations indicate that NPGPA1 is probably a single-copy gene, whereas at least two copies of NPGPB1 exist in the N. plumbaginifolia genome. Northern analyses reveal that both NPGPA1 and NPGPB1 mRNA are expressed in all embryogenic stages and plant tissues examined and their expression is obviously regulated by the plant hormone auxin. Immunohistological localization of NPGPalpha1 and NPGPbeta1 preferentially on plasma and endoplasmic reticulum membranes and their immunochemical detection exclusively in microsomal cell fractions implicate membrane association of both proteins. The temporal and spatial expression patterns of NPGPA1 and NPGPB1 show conformity as well as differences. This could account for not only cooperative, but also individual activities of both subunits during embryogenesis and plant development.

Sweet Potato [Ipomoea batatas (L.) Lam].

PubMed

Song, Guo-qing; Yamaguchi, Ken-ichi

2006-01-01

Among the available transformation methods reported on sweet potato, Agrobacterium tumefaciens-mediated transformation is more successful and desirable. Stem explants have shown to be ideal for the transformation of sweet potato because of their ready availability as explants, the simple transformation process, and high-frequency-regeneration via somatic embryogenesis. Under the two-step kanamycin-hygromycin selection method and using the appropriate explants type (stem explants), the efficiency of transformation can be considerably improved in cv. Beniazuma. The high efficiency in the transformation of stem explants suggests that the transformation protocol described in this chapter warrants testing for routine stable transformation of diverse varieties of sweet potato.

Micropropagation systems of Feijoa (Acca sellowiana (O. Berg) Burret).

PubMed

Guerra, Miguel Pedro; Cangahuala-Inocente, Gabriela Claudia; Vesco, Lirio Luiz Dal; Pescador, Rosete; Caprestano, Clarissa Alves

2013-01-01

Acca sellowiana (O. Berg) Burret sin. Feijoa sellowiana (Myrtaceae) is a semiwoody fruit species native to South Brazil, Uruguay, and Argentina; edible fruits are tasty. The naturally occurring populations in Santa Catarina State show high variability in fruit size, color, and other features. A breeding program launched in 1990 resulted in the release of four Brazilian commercial varieties. The conventional clonal propagation methods of this species, such as cutting and grafting, have shown low efficiency. Therefore, tissue culture techniques were developed for mass propagation. This chapter describes several protocols based on organogenesis and somatic embryogenesis. Additional techniques including synthetic seed technology and temporary immersion system are also described.

Analysis of the global transcriptome of longan (Dimocarpus longan Lour.) embryogenic callus using Illumina paired-end sequencing

PubMed Central

2013-01-01

Background Longan is a tropical/subtropical fruit tree of great economic importance in Southeast Asia. Progress in understanding molecular mechanisms of longan embryogenesis, which is the primary influence on fruit quality and yield, is slowed by lack of transcriptomic and genomic information. Illumina second generation sequencing, which is suitable for generating enormous numbers of transcript sequences that can be used for functional genomic analysis of longan. Results In this study, a longan embryogenic callus (EC) cDNA library was sequenced using an Illumina HiSeq 2000 system. A total of 64,876,258 clean reads comprising 5.84 Gb of nucleotides were assembled into 68,925 unigenes of 448-bp mean length, with unigenes ≥1000 bp accounting for 8.26% of the total. Using BLASTx, 40,634 unigenes were found to have significant similarity with accessions in Nr and Swiss- Prot databases. Of these, 38,845 unigenes were assigned to 43 GO sub-categories and 17,118 unigenes were classified into 25 COG sub-groups. In addition, 17,306 unigenes mapped to 199 KEGG pathways, with the categories of Metabolic pathways, Plant-pathogen interaction, Biosynthesis of secondary metabolites, and Genetic information processing being well represented. Analyses of unigenes ≥1000 bp revealed 328 embryogenesis-related unigenes as well as numerous unigenes expressed in EC associated with functions of reproductive growth, such as flowering, gametophytogenesis, and fertility, and vegetative growth, such as root and shoot growth. Furthermore, 23 unigenes related to embryogenesis and reproductive and vegetative growth were validated by quantitative real time PCR (qPCR) in samples from different stages of longan somatic embryogenesis (SE); their differentially expressions in the various embryogenic cultures indicated their possible roles in longan SE. Conclusions The quantity and variety of expressed EC genes identified in this study is sufficient to serve as a global transcriptome dataset for longan EC and to provide more molecular resources for longan functional genomics. PMID:23957614

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

PubMed

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

2015-01-01

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

Exome sequencing of bilateral testicular germ cell tumors suggests independent development lineages.

PubMed

Brabrand, Sigmund; Johannessen, Bjarne; Axcrona, Ulrika; Kraggerud, Sigrid M; Berg, Kaja G; Bakken, Anne C; Bruun, Jarle; Fosså, Sophie D; Lothe, Ragnhild A; Lehne, Gustav; Skotheim, Rolf I

2015-02-01

Intratubular germ cell neoplasia, the precursor of testicular germ cell tumors (TGCTs), is hypothesized to arise during embryogenesis from developmentally arrested primordial germ cells (PGCs) or gonocytes. In early embryonal life, the PGCs migrate from the yolk sac to the dorsal body wall where the cell population separates before colonizing the genital ridges. However, whether the malignant transformation takes place before or after this separation is controversial. We have explored the somatic exome-wide mutational spectra of bilateral TGCT to provide novel insight into the in utero critical time frame of malignant transformation and TGCT pathogenesis. Exome sequencing was performed in five patients with bilateral TGCT (eight tumors), of these three patients in whom both tumors were available (six tumors) and two patients each with only one available tumor (two tumors). Selected loci were explored by Sanger sequencing in 71 patients with bilateral TGCT. From the exome-wide mutational spectra, no identical mutations in any of the three bilateral tumor pairs were identified. Exome sequencing of all eight tumors revealed 87 somatic non-synonymous mutations (median 10 per tumor; range 5-21), some in already known cancer genes such as CIITA, NEB, platelet-derived growth factor receptor α (PDGFRA), and WHSC1. SUPT6H was found recurrently mutated in two tumors. We suggest independent development lineages of bilateral TGCT. Thus, malignant transformation into intratubular germ cell neoplasia is likely to occur after the migration of PGCs. We reveal possible drivers of TGCT pathogenesis, such as mutated PDGFRA, potentially with therapeutic implications for TGCT patients. Copyright © 2014 Neoplasia Press, Inc. Published by Elsevier Inc. All rights reserved.

Carry-over effects modulated by salinity during the early ontogeny of the euryhaline crab Hemigrapsus crenulatus from the Southeastern Pacific coast: Development time and carbon and energy content of offspring.

PubMed

Urzúa, Ángel; Bascur, Miguel; Guzmán, Fabián; Urbina, Mauricio

2018-03-01

Hemigrapsus crenulatus is a key species of coastal and estuarine ecosystems in the Southeastern Pacific and New Zealand. Since the gravid females-and their embryos-develop under conditions of variable salinity, we propose that low external salinity will be met with an increase in energy expenditures in order to maintain osmoregulation; subsequently, the use of energy reserves for reproduction will be affected. In this study, we investigate in H. crenulatus whether 1) the biomass and energy content of embryos is influenced by salinity experienced during oogenesis and embryogenesis and 2) how variation in the biomass and energy content of embryos affects larval energetic condition at hatching. Here at low salinity (5PSU), egg-bearing females experienced massive and frequent egg losses, and therefore the development of their eggs during embryogenesis was not completed. In turn, at intermediate and high salinity (15 and 30PSU) embryogenesis was completed, egg development was successful, and larvae were obtained. Consistently, larvae hatched from eggs produced and incubated at high salinity (30PSU) were larger, had higher dry weight, and had increased carbon content and energy than larvae hatched from eggs produced at intermediate salinity (15PSU). From these results, it is seen that the size and biomass of early life stages of H. crenulatus can be affected by environmental salinity experienced during oogenesis and embryogenesis, and this variation can then directly affect the energetic condition of offspring at birth. Therefore, this study reveals a "cascade effect" modulated by salinity during the early ontogeny. Copyright © 2018 Elsevier Inc. All rights reserved.

Cloning and analysis of fetal ovary microRNAs in cattle.

PubMed

Tripurani, Swamy K; Xiao, Caide; Salem, Mohamed; Yao, Jianbo

2010-07-01

Ovarian folliculogenesis and early embryogenesis are complex processes, which require tightly regulated expression and interaction of a multitude of genes. Small endogenous RNA molecules, termed microRNAs (miRNAs), are involved in the regulation of gene expression during folliculogenesis and early embryonic development. To identify miRNAs in bovine oocytes/ovaries, a bovine fetal ovary miRNA library was constructed. Sequence analysis of random clones from the library identified 679 miRNA sequences, which represent 58 distinct bovine miRNAs. Of these distinct miRNAs, 42 are known bovine miRNAs present in the miRBase database and the remaining 16 miRNAs include 15 new bovine miRNAs that are homologous to miRNAs identified in other species, and one novel miRNA, which does not match any miRNAs in the database. The precursor sequences for 14 of the new 15 miRNAs as well as the novel miRNA were identified from the bovine genome database and their hairpin structures were predicted. Expression analysis of the 58 miRNAs in fetal ovaries in comparison to somatic tissue pools identified 8 miRNAs predominantly expressed in fetal ovaries. Further analysis of the eight miRNAs in germinal vesicle (GV) stage oocytes identified two miRNAs (bta-mir424 and bta-mir-10b), that are highly abundant in GV oocytes. Both miRNAs show similar expression patterns during oocyte maturation and preimplantation development of bovine embryos, being abundant in GV and MII stage oocytes, as well as in early stage embryos (until 16-cell stage). The amount of the novel miRNA is relatively small in oocytes and early cleavage embryos but greater in blastocysts, suggesting a role of this miRNA in blastocyst cell differentiation. Copyright 2010 Elsevier B.V. All rights reserved.

Sex-specific impact of early-life adversity on chronic pain: a large population-based study in Japan.

PubMed

Yamada, Keiko; Matsudaira, Ko; Tanaka, Eizaburo; Oka, Hiroyuki; Katsuhira, Junji; Iso, Hiroyasu

2017-01-01

Responses to early-life adversity may differ by sex. We investigated the sex-specific impact of early-life adversity on chronic pain, chronic multisite pain, and somatizing tendency with chronic pain. We examined 4229 respondents aged 20-79 years who participated in the Pain Associated Cross-Sectional Epidemiological Survey in Japan. Outcomes were: 1) chronic pain prevalence, 2) multisite pain (≥3 sites) prevalence, and 3) multiple somatic symptoms (≥3 symptoms) among respondents with chronic pain related to the presence or absence of early-life adversity. Multivariable-adjusted odds ratios (ORs) were calculated with 95% confidence intervals using a logistic regression model including age, smoking status, exercise routine, sleep time, body mass index, household expenditure, and the full distribution of scores on the Mental Health Inventory-5. We further adjusted for pain intensity when we analyzed the data for respondents with chronic pain. The prevalence of chronic pain was higher among respondents reporting the presence of early-life adversity compared with those reporting its absence, with multivariable ORs of 1.62 (1.22-2.15, p <0.01) in men and 1.47 (1.13-1.90, p <0.01) in women. Among women with chronic pain, early-life adversity was associated with multisite pain and multiple somatic symptoms; multivariable ORs were 1.78 (1.22-2.60, p <0.01) for multisite pain and 1.89 (1.27-2.83, p <0.01) for ≥3 somatic symptoms. No associations were observed between early-life adversity and chronic multisite pain or multiple somatic symptoms among men with chronic pain. Early-life adversity may be linked to a higher prevalence of chronic pain among both sexes and to multisite pain and somatizing tendency among women with chronic pain.

Drosophila embryogenesis scales uniformly across temperature in developmentally diverse species.

PubMed

Kuntz, Steven G; Eisen, Michael B

2014-04-01

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 [Formula: see text]33 hours at 17.5 °C, and accelerates with increasing temperature to a low of 16 hours at 27.5 °C, above which embryogenesis slows slightly. Remarkably, while the total time of embryogenesis varies over two fold, the relative timing of events from cellularization through hatching is constant across temperatures. To further explore the relationship between temperature and embryogenesis, we expanded our analysis to cover ten additional Drosophila species of varying climatic origins. Six of these species, like D. melanogaster, are of tropical origin, and embryogenesis time at different temperatures was similar for them all. D. mojavensis, a sub-tropical fly, develops slower than the tropical species at lower temperatures, while D. virilis, a temperate fly, exhibits slower development at all temperatures. The alpine sister species D. persimilis and D. pseudoobscura develop as rapidly as tropical flies at cooler temperatures, but exhibit diminished acceleration above 22.5 °C and have drastically slowed development by 30 °C. Despite ranging from 13 hours for D. erecta at 30 °C to 46 hours for D. virilis at 17.5 °C, the relative timing of events from cellularization through hatching is constant across all species and temperatures examined here, suggesting the existence of a previously unrecognized timer controlling the progress of embryogenesis that has been tuned by natural selection as each species diverges.

Cryopreservation of somatic embryogenic cultures of Pinus pinaster: effects on regrowth and embryo maturation.

PubMed

Álvarez, José M; Cortizo, Millán; Ordás, Ricardo J

2012-01-01

Pinus pinaster is one of the most economically important conifers in the world. Somatic embryogenesis is a powerful tool in breeding programmes because it allows the generation of a great number of different clonal lines from seeds of superior genotypes. Unfortunately, embryogenic competence decreases with the age of cultures. Therefore, it is necessary to have a cryopreservation protocol that ensures a continuous supply of juvenile mass while allowing good maturation and conversion rates into vigorously growing plants. In this work we studied the influence of several cryopreservation parameters, such as cryoprotectant solution and pre-cooling temperature, on embryogenic culture regrowth and embryo maturation. Recovery of rewarmed samples after cryopreservation in a -150 degree C freezer depended on the cooling temperature reached prior to plunging the tubes into liquid nitrogen. As a result, we present an optimised cryopreservation protocol that ensures high recovery and embryo maturation rates. The protocol presented is a simple and fast alternative and enabled successful cryopreservation and recovery of 100 percent of the lines tested. Cryopreserved lines presented the same maturation rates as non-cryopreserved controls.

Controlled insertional mutagenesis using a LINE-1 (ORFeus) gene-trap mouse model.

PubMed

O'Donnell, Kathryn A; An, Wenfeng; Schrum, Christina T; Wheelan, Sarah J; Boeke, Jef D

2013-07-16

A codon-optimized mouse LINE-1 element, ORFeus, exhibits dramatically higher retrotransposition frequencies compared with its native long interspersed element 1 counterpart. To establish a retrotransposon-mediated mouse model with regulatable and potent mutagenic capabilities, we generated a tetracycline (tet)-regulated ORFeus element harboring a gene-trap cassette. Here, we show that mice expressing tet-ORFeus broadly exhibit robust retrotransposition in somatic tissues when treated with doxycycline. Consistent with a significant mutagenic burden, we observed a reduced number of double transgenic animals when treated with high-level doxycycline during embryogenesis. Transgene induction in skin resulted in a white spotting phenotype due to somatic ORFeus-mediated mutations that likely disrupt melanocyte development. The data suggest a high level of transposition in melanocyte precursors and consequent mutation of genes important for melanoblast proliferation, differentiation, or migration. These findings reveal the utility of a retrotransposon-based mutagenesis system as an alternative to existing DNA transposon systems. Moreover, breeding these mice to different tet-transactivator/reversible tet-transactivator lines supports broad functionality of tet-ORFeus because of the potential for dose-dependent, tissue-specific, and temporal-specific mutagenesis.

DND protein functions as a translation repressor during zebrafish embryogenesis.

PubMed

Kobayashi, Manami; Tani-Matsuhana, Saori; Ohkawa, Yasuka; Sakamoto, Hiroshi; Inoue, Kunio

2017-03-04

Germline and somatic cell distinction is regulated through a combination of microRNA and germ cell-specific RNA-binding proteins in zebrafish. An RNA-binding protein, DND, has been reported to relieve the miR-430-mediated repression of some germ plasm mRNAs such as nanos3 and tdrd7 in primordial germ cells (PGCs). Here, we showed that miR-430-mediated repression is not counteracted by the overexpression of DND protein in somatic cells. Using a λN-box B tethering assay in the embryo, we found that tethering of DND to reporter mRNA results in translation repression without affecting mRNA stability. Translation repression by DND was not dependent on another germline-specific translation repressor, Nanos3, in zebrafish embryos. Moreover, our data suggested that DND represses translation of nanog and dnd mRNAs, whereas an RNA-binding protein DAZ-like (DAZL) promotes dnd mRNA translation. Thus, our study showed that DND protein functions as a translation repressor of specific mRNAs to control PGC development in zebrafish. Copyright © 2017 Elsevier Inc. All rights reserved.

Essential role of the TFIID subunit TAF4 in murine embryogenesis and embryonic stem cell differentiation

PubMed Central

Langer, Diana; Martianov, Igor; Alpern, Daniel; Rhinn, Muriel; Keime, Céline; Dollé, Pascal; Mengus, Gabrielle; Davidson, Irwin

2016-01-01

TAF4 (TATA-binding protein-associated factor 4) and its paralogue TAF4b are components of the TFIID core module. We inactivated the murine Taf4a gene to address Taf4 function during embryogenesis. Here we show that Taf4a−/− embryos survive until E9.5 where primary germ layers and many embryonic structures are identified showing Taf4 is dispensable for their specification. In contrast, Taf4 is required for correct patterning of the trunk and anterior structures, ventral morphogenesis and proper heart positioning. Overlapping expression of Taf4a and Taf4b during embryogenesis suggests their redundancy at early stages. In agreement with this, Taf4a−/− embryonic stem cells (ESCs) are viable and comprise Taf4b-containing TFIID. Nevertheless, Taf4a−/− ESCs do not complete differentiation into glutamatergic neurons and cardiomyocytes in vitro due to impaired preinitiation complex formation at the promoters of critical differentiation genes. We define an essential role of a core TFIID TAF in differentiation events during mammalian embryogenesis. PMID:27026076

Essential role of the TFIID subunit TAF4 in murine embryogenesis and embryonic stem cell differentiation.

PubMed

Langer, Diana; Martianov, Igor; Alpern, Daniel; Rhinn, Muriel; Keime, Céline; Dollé, Pascal; Mengus, Gabrielle; Davidson, Irwin

2016-03-30

TAF4 (TATA-binding protein-associated factor 4) and its paralogue TAF4b are components of the TFIID core module. We inactivated the murine Taf4a gene to address Taf4 function during embryogenesis. Here we show that Taf4a(-/-) embryos survive until E9.5 where primary germ layers and many embryonic structures are identified showing Taf4 is dispensable for their specification. In contrast, Taf4 is required for correct patterning of the trunk and anterior structures, ventral morphogenesis and proper heart positioning. Overlapping expression of Taf4a and Taf4b during embryogenesis suggests their redundancy at early stages. In agreement with this, Taf4a(-/-) embryonic stem cells (ESCs) are viable and comprise Taf4b-containing TFIID. Nevertheless, Taf4a(-/-) ESCs do not complete differentiation into glutamatergic neurons and cardiomyocytes in vitro due to impaired preinitiation complex formation at the promoters of critical differentiation genes. We define an essential role of a core TFIID TAF in differentiation events during mammalian embryogenesis.

Dual mode of embryonic development is highlighted by expression and function of Nasonia pair-rule genes

PubMed Central

Rosenberg, Miriam I; Brent, Ava E; Payre, François; Desplan, Claude

2014-01-01

Embryonic anterior–posterior patterning is well understood in Drosophila, which uses ‘long germ’ embryogenesis, in which all segments are patterned before cellularization. In contrast, most insects use ‘short germ’ embryogenesis, wherein only head and thorax are patterned in a syncytial environment while the remainder of the embryo is generated after cellularization. We use the wasp Nasonia (Nv) to address how the transition from short to long germ embryogenesis occurred. Maternal and gap gene expression in Nasonia suggest long germ embryogenesis. However, the Nasonia pair-rule genes even-skipped, odd-skipped, runt and hairy are all expressed as early blastoderm pair-rule stripes and late-forming posterior stripes. Knockdown of Nv eve, odd or h causes loss of alternate segments at the anterior and complete loss of abdominal segments. We propose that Nasonia uses a mixed mode of segmentation wherein pair-rule genes pattern the embryo in a manner resembling Drosophila at the anterior and ancestral Tribolium at the posterior. DOI: http://dx.doi.org/10.7554/eLife.01440.001 PMID:24599282

Improvement of tissue culture, genetic transformation, and applications of biotechnology to Brassica.

PubMed

Ravanfar, Seyed Ali; Orbovic, Vladimir; Moradpour, Mahdi; Abdul Aziz, Maheran; Karan, Ratna; Wallace, Simon; Parajuli, Saroj

2017-04-01

Development of in vitro plant regeneration method from Brassica explants via organogenesis and somatic embryogenesis is influenced by many factors such as culture environment, culture medium composition, explant sources, and genotypes which are reviewed in this study. An efficient in vitro regeneration system to allow genetic transformation of Brassica is a crucial tool for improving its economical value. Methods to optimize transformation protocols for the efficient introduction of desirable traits, and a comparative analysis of these methods are also reviewed. Hence, binary vectors, selectable marker genes, minimum inhibitory concentration of selection agents, reporter marker genes, preculture media, Agrobacterium concentration and regeneration ability of putative transformants for improvement of Agrobacterium-mediated transformation of Brassica are discussed.

Finger millet [Eleusine coracana (L.) Gaertn].

PubMed

Ceasar, Stanislaus Antony; Ignacimuthu, Savarimuthu

2015-01-01

Millets are the primary food source for millions of people in tropical regions of the world supplying mineral nutrition and protein. In this chapter, we describe an optimized protocol for the Agrobacterium-mediated transformation of finger millet variety GPU 45. Agrobacterium strain LBA4404 harboring plasmid pCAMBIA1301 which contains hygromycin phosphotransferase (hph) as selectable marker gene and β-glucuronidase (GUS) as reporter gene has been used. This protocol utilizes the shoot apex explants for the somatic embryogenesis and regeneration of finger millet after the transformation by Agrobacterium. Desiccation of explants during cocultivation helps for the better recovery of transgenic plants. This protocol is very useful for the efficient production of transgenic plants in finger millet through Agrobacterium-mediated transformation.

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

PubMed Central

2013-01-01

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

Notch1 is asymmetrically distributed from the beginning of embryogenesis and controls the ventral center.

PubMed

Castro Colabianchi, Aitana M; Revinski, Diego R; Encinas, Paula I; Baez, María Verónica; Monti, Renato J; Abinal, Mateo Rodríguez; Kodjabachian, Laurent; Franchini, Lucía F; López, Silvia L

2018-06-04

Based on functional evidence, we have previously demonstrated that an early ventral Notch1 activity restricts dorsoanterior development in Xenopus We found that Notch1 has ventralizing properties and abolishes the dorsalizing activity of β-catenin by reducing its steady state levels, in a process that does not require β-catenin phosphorylation by glycogen synthase kinase-3β. In the present work, we demonstrate that Notch1 mRNA and protein are enriched in the ventral region from the beginning of the embryogenesis in Xenopus This is the earliest sign of ventral development, preceding the localized expression of wnt8a , bmp4 and ventxs genes in the ventral center and the dorsal accumulation of nuclear β-catenin. Knock-down experiments indicate that Notch1 is necessary for the normal expression of genes essential for ventral-posterior development. These results indicate that during early embryogenesis, ventrally located Notch1 promotes the development of the ventral center. Together with our previous evidence, these results suggest that ventral enrichment of Notch1 underlies the process by which Notch1 participates in restricting nuclear accumulation of β-catenin to the dorsal side. © 2018. Published by The Company of Biologists Ltd.

The near demise and subsequent revival of classical genetics for investigating Caenorhabditis elegans embryogenesis: RNAi meets next-generation DNA sequencing.

PubMed

Bowerman, Bruce

2011-10-01

Molecular genetic investigation of the early Caenorhabditis elegans embryo has contributed substantially to the discovery and general understanding of the genes, pathways, and mechanisms that regulate and execute developmental and cell biological processes. Initially, worm geneticists relied exclusively on a classical genetics approach, isolating mutants with interesting phenotypes after mutagenesis and then determining the identity of the affected genes. Subsequently, the discovery of RNA interference (RNAi) led to a much greater reliance on a reverse genetics approach: reducing the function of known genes with RNAi and then observing the phenotypic consequences. Now the advent of next-generation DNA sequencing technologies and the ensuing ease and affordability of whole-genome sequencing are reviving the use of classical genetics to investigate early C. elegans embryogenesis.

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

Generation of autotetraploid plant of ginger (Zingiber officinale Rosc.) and its quality evaluation

PubMed Central

Kun-Hua, Wei; Jian-Hua, Miao; He-Ping, Huang; Shan-Lin, Gao

2011-01-01

Background: Zingiber officinale Rosc. is not only an important medical plant in China, but also one of the most commonly used plant spices around the world. Early researches in Z. officinale Rosc. were focused on rapid propagation, germplasm preservation, and somatic embryogenesis, only a few reports focused on the generation of tetraploid ginger plants with colchicines treatment in vitro. Materials and Methods: The adventitious buds were submerged into different concentrations of colchicine water solution for different time to induce polyploid plants, and the induced buds were identified by root-tip chromosome determination and stomatal apparatus observation. Eighteen selected tetraploid lines were transferred to the field, and the leaf characteristics, rhizome yield, contents of volatile oil and gingerol were respectively evaluated to provide evidence of high-yield and good qualities of tetraploid ginger. Results: The induction rate reached as high as 33.3% of treated buds. More than 48 lines of autotetraploid plants were obtained. All tetraploid plants showed typical polyploidy characteristics. All of the 18 selected tetraploid lines possessed higher rhizome yield and overall productivity of volatile oil and gingerol than those of the control. Conclusion: Five elite lines have been selected for further selection and breeding new varieties for commercial production in agricultural production. PMID:21969790

Evolution of early embryogenesis in rhabditid nematodes

PubMed Central

Brauchle, Michael; Kiontke, Karin; MacMenamin, Philip; Fitch, David H. A.; Piano, Fabio

2009-01-01

The cell biological events that guide early embryonic development occur with great precision within species but can be quite diverse across species. How these cellular processes evolve and which molecular components underlie evolutionary changes is poorly understood. To begin to address these questions, we systematically investigated early embryogenesis, from the one- to the four-cell embryo, in 34 nematode species related to C. elegans. We found 40 cell-biological characters that captured the phenotypic differences between these species. By tracing the evolutionary changes on a molecular phylogeny, we found that these characters evolved multiple times and independently of one another. Strikingly, all these phenotypes are mimicked by single-gene RNAi experiments in C. elegans. We use these comparisons to hypothesize the molecular mechanisms underlying the evolutionary changes. For example, we predict that a cell polarity module was altered during the evolution of the Protorhabditis group and show that PAR-1, a kinase localized asymmetrically in C. elegans early embryos, is symmetrically localized in the one-cell stage of Protorhabditis group species. Our genome-wide approach identifies candidate molecules—and thereby modules—associated with evolutionary changes in cell-biological phenotypes. PMID:19643102

The mRNA-bound proteome of the early fly embryo

PubMed Central

Wessels, Hans-Hermann; Imami, Koshi; Baltz, Alexander G.; Kolinski, Marcin; Beldovskaya, Anastasia; Selbach, Matthias; Small, Stephen; Ohler, Uwe; Landthaler, Markus

2016-01-01

Early embryogenesis is characterized by the maternal to zygotic transition (MZT), in which maternally deposited messenger RNAs are degraded while zygotic transcription begins. Before the MZT, post-transcriptional gene regulation by RNA-binding proteins (RBPs) is the dominant force in embryo patterning. We used two mRNA interactome capture methods to identify RBPs bound to polyadenylated transcripts within the first 2 h of Drosophila melanogaster embryogenesis. We identified a high-confidence set of 476 putative RBPs and confirmed RNA-binding activities for most of 24 tested candidates. Most proteins in the interactome are known RBPs or harbor canonical RBP features, but 99 exhibited previously uncharacterized RNA-binding activity. mRNA-bound RBPs and TFs exhibit distinct expression dynamics, in which the newly identified RBPs dominate the first 2 h of embryonic development. Integrating our resource with in situ hybridization data from existing databases showed that mRNAs encoding RBPs are enriched in posterior regions of the early embryo, suggesting their general importance in posterior patterning and germ cell maturation. PMID:27197210

Genome-Wide Mapping of Collier In Vivo Binding Sites Highlights Its Hierarchical Position in Different Transcription Regulatory Networks

PubMed Central

Dubois, Laurence; Bataillé, Laetitia; Painset, Anaïs; Le Gras, Stéphanie; Jost, Bernard; Crozatier, Michèle; Vincent, Alain

2015-01-01

Collier, the single Drosophila COE (Collier/EBF/Olf-1) transcription factor, is required in several developmental processes, including head patterning and specification of muscle and neuron identity during embryogenesis. To identify direct Collier (Col) targets in different cell types, we used ChIP-seq to map Col binding sites throughout the genome, at mid-embryogenesis. In vivo Col binding peaks were associated to 415 potential direct target genes. Gene Ontology analysis revealed a strong enrichment in proteins with DNA binding and/or transcription-regulatory properties. Characterization of a selection of candidates, using transgenic CRM-reporter assays, identified direct Col targets in dorso-lateral somatic muscles and specific neuron types in the central nervous system. These data brought new evidence that Col direct control of the expression of the transcription regulators apterous and eyes-absent (eya) is critical to specifying neuronal identities. They also showed that cross-regulation between col and eya in muscle progenitor cells is required for specification of muscle identity, revealing a new parallel between the myogenic regulatory networks operating in Drosophila and vertebrates. Col regulation of eya, both in specific muscle and neuronal lineages, may illustrate one mechanism behind the evolutionary diversification of Col biological roles. PMID:26204530

The effect of temperature and light on embryogenesis and post-embryogenesis of the spider Eratigena atrica (Araneae, Agelenidae).

PubMed

Napiórkowska, Teresa; Kobak, Jarosław; Napiórkowski, Paweł; Templin, Julita

2018-02-01

Embryogenesis and post-embryogenesis of spiders depend on several environmental factors including light and temperature. This study was aimed at evaluating the impact of different thermal and lighting conditions on embryonic and early post-embryonic development of Eratigena atrica. Embryos, larvae, nymphs I and II were incubated at constant temperatures of 12, 22, 25 and 32°C under three different light regimes: light, dark, light/dark. Extreme temperatures (12 and 32°C) significantly increased mortality of embryos (to 100%) and nymphs II, whereas larvae and nymphs I suffered reduced survival only at the lowest temperature. Moreover, the lowest temperature reduced the development rate of all stages. The impact of light conditions was less pronounced and more variable: constant light reduced the survival of nymphs I at lower temperatures, but increased that of larvae. Moreover, light increased the time of embryonic development and duration of nymphal stages, particularly at lower temperatures (12-22°C). Thus, the most optimal locations for spiders seem to be dark (though except larval stage) and warm (25°C) sites, where their development is fastest and mortality lowest. Copyright © 2017 Elsevier Ltd. All rights reserved.

Arabidopsis thaliana Somatic Embryogenesis Receptor Kinase 1 protein is present in sporophytic and gametophytic cells and undergoes endocytosis.

PubMed

Kwaaitaal, M A C J; de Vries, S C; Russinova, E

2005-10-01

Arabidopsis thaliana plants expressing AtSERK1 fused to yellow-fluorescent protein were generated. Fluorescence was detected predominantly at the cell periphery, most likely the plasma membrane, of cells in ovules, embryo sacs, anthers, and embryos and in seedlings. The AtSERK1 protein was detected in diverse cell types including the epidermis and the vascular bundles. In some cells, fluorescent receptors were seen in small vesicle-like compartments. After application of the fungal toxin Brefeldin A, the fluorescent receptors were rapidly internalized in the root meristem and root vascular tissue. We conclude that the AtSERK1 receptor functions in a common signalling pathway employed in both sporophytic and gametophytic cells.

Protocols for Improvement of Black Pepper (Piper nigrum L.) Utilizing Biotechnological Tools.

PubMed

Nirmal Babu, K; Divakaran, Minoo; Yamuna, G; Ravindran, P N; Peter, K V

2016-01-01

Black pepper, Piper nigrum L., the "King of spices" is the most widely used spice growing in the South-Western region of India. The humid tropical evergreen forest bordering the Malabar Coast (Western Ghats is one of the hot spot areas of plant bio-diversity on earth) is its center of origin and diversity. However, the crop faces constraints like rampant fungal and viral diseases, lack of disease free planting material, hence biotechnological tools can be utilized to address these problems and strides have been made successfully. The standardization of micropropagation, somatic embryogenesis, in vitro conservation, protoplast isolation, and genetic transformation protocols are described here. The protocols could be utilized to achieve similar goals in the related species of Piper too.

Breakthrough in chloroplast genetic engineering of agronomically important crops

PubMed Central

Daniell, Henry; Kumar, Shashi; Dufourmantel, Nathalie

2012-01-01

Chloroplast genetic engineering offers several unique advantages, including high-level transgene expression, multi-gene engineering in a single transformation event and transgene containment by maternal inheritance, as well as a lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. More than 40 transgenes have been stably integrated and expressed using the tobacco chloroplast genome to confer desired agronomic traits or express high levels of vaccine antigens and biopharmaceuticals. Despite such significant progress, this technology has not been extended to major crops. However, highly efficient soybean, carrot and cotton plastid transformation has recently been accomplished through somatic embryogenesis using species-specific chloroplast vectors. This review focuses on recent exciting developments in this field and offers directions for further research and development. PMID:15866001

Nucleostemin Delays Cellular Senescence and Negatively Regulates TRF1 Protein Stability▿ †

PubMed Central

Zhu, Qubo; Yasumoto, Hiroaki; Tsai, Robert Y. L.

2006-01-01

Nucleostemin (NS) encodes a nucleolar GTP-binding protein highly enriched in the stem cells and cancer cells. To determine its biological activity in vivo, we generated NS loss- and gain-of-function mouse models. The embryogenesis of homozygous NS-null (NS−/−) mice was aborted before the blastula stage. Although the growth and fertility of heterozygous NS-null (NS+/−) mice appeared normal, NS+/− mouse embryonic fibroblasts (MEFs) had fewer NS proteins, a lower population growth rate, and higher percentages of senescent cells from passage 5 (P5) to P7 than their wild-type littermates. Conversely, transgenic overexpression of NS could rescue the NS−/− embryo in a dose-dependent manner, increase the population growth rate, and reduce the senescent percentage of MEFs. Cell cycle analyses revealed increased pre-G1 percentages in the late-passage NS+/− MEF cultures compared to the wild-type cultures. We demonstrated that NS could interact with telomeric repeat-binding factor 1 (TRF1) and enhance the degradation but not the ubiquitination of the TRF1 protein, which negatively regulates telomere length and is essential for early embryogenesis. This work demonstrates the roles of NS in establishing early embryogenesis and delaying cellular senescence of MEFs and reveals a mechanism of a NS-regulated degradation of TRF1. PMID:17000763

Telomere length regulation during cloning, embryogenesis and ageing.

PubMed

Schaetzlein, S; Rudolph, K L

2005-01-01

Telomeres are nucleoprotein complexes at the end of eukaryotic chromosomes with an essential role in chromosome capping. Owing to the end-replication problem of DNA polymerase, telomeres shorten during each cell division. When telomeres become critically short, they loose their capping function, which in turn induces a DNA damage-like response. This mechanism inhibits cell proliferation at the senescence stage and there is evidence that it limits the regenerative capacity of tissues and organs during chronic diseases and ageing. The holoenzyme telomerase synthesises telomeric DNA de novo, but, in humans, it is active only during embryogenesis, in immature germ cells and in a subset of stem/progenitor cells during postnatal life. Telomere length can be maintained or increased by telomerase, a process that appears to be regulated by a variety of telomere-binding proteins that control telomerase recruitment and activity at the telomeres. During embryogenesis, telomerase is strongly activated at the morula/blastocyst transition. At this transition, telomeres are significantly elongated in murine and bovine embryos. Early embryonic telomere elongation is telomerase dependent and leads to a rejuvenation of telomeres in cloned bovine embryos. Understanding of the molecular mechanisms underlying this early embryonic telomere elongation programme is of great interest for medical research in the fields of regeneration, cell therapies and therapeutic cloning.

Nitric oxide synthase during early embryonic development in silkworm Bombyx mori: Gene expression, enzyme activity, and tissue distribution.

PubMed

Kitta, Ryo; Kuwamoto, Marina; Yamahama, Yumi; Mase, Keisuke; Sawada, Hiroshi

2016-12-01

To elucidate the mechanism for embryonic diapause or the breakdown of diapause in Bombyx mori, we biochemically analyzed nitric oxide synthase (NOS) during the embryogenesis of B. mori. The gene expression and enzyme activity of B. mori NOS (BmNOS) were examined in diapause, non-diapause, and HCl-treated diapause eggs. In the case of HCl-treated diapause eggs, the gene expression and enzyme activity of BmNOS were induced by HCl treatment. However, in the case of diapause and non-diapause eggs during embryogenesis, changes in the BmNOS activity and gene expressions did not coincide except 48-60 h after oviposition in diapause eggs. The results imply that changes in BmNOS activity during the embryogenesis of diapause and non-diapause eggs are regulated not only at the level of transcription but also post-transcription. The distribution and localization of BmNOS were also investigated with an immunohistochemical technique using antibodies against the universal NOS; the localization of BmNOS was observed mainly in the cytoplasm of yolk cells in diapause eggs and HCl-treated diapause eggs. These data suggest that BmNOS has an important role in the early embryonic development of the B. mori. © 2016 Japanese Society of Developmental Biologists.

Analysis of Large Seeds from Three Different Medicago truncatula Ecotypes Reveals a Potential Role of Hormonal Balance in Final Size Determination of Legume Grains

PubMed Central

Bandyopadhyay, Kaustav; Uluçay, Orhan; Şakiroğlu, Muhammet; Udvardi, Michael K.; Verdier, Jerome

2016-01-01

Legume seeds are important as protein and oil source for human diet. Understanding how their final seed size is determined is crucial to improve crop yield. In this study, we analyzed seed development of three accessions of the model legume, Medicago truncatula, displaying contrasted seed size. By comparing two large seed accessions to the reference accession A17, we described mechanisms associated with large seed size determination and potential factors modulating the final seed size. We observed that early events during embryogenesis had a major impact on final seed size and a delayed heart stage embryo development resulted to large seeds. We also observed that the difference in seed growth rate was mainly due to a difference in embryo cell number, implicating a role of cell division rate. Large seed accessions could be explained by an extended period of cell division due to a longer embryogenesis phase. According to our observations and recent reports, we observed that auxin (IAA) and abscisic acid (ABA) ratio could be a key determinant of cell division regulation at the end of embryogenesis. Overall, our study highlights that timing of events occurring during early seed development play decisive role for final seed size determination. PMID:27618017

Small RNA profiling and degradome analysis reveal regulation of microRNA in peanut embryogenesis and early pod development.

PubMed

Gao, Chao; Wang, Pengfei; Zhao, Shuzhen; Zhao, Chuanzhi; Xia, Han; Hou, Lei; Ju, Zheng; Zhang, Ye; Li, Changsheng; Wang, Xingjun

2017-03-02

As a typical geocarpic plant, peanut embryogenesis and pod development are complex processes involving many gene regulatory pathways and controlled by appropriate hormone level. MicroRNAs (miRNAs) are small non-coding RNAs that play indispensable roles in post-transcriptional gene regulation. Recently, identification and characterization of peanut miRNAs has been described. However, whether miRNAs participate in the regulation of peanut embryogenesis and pod development has yet to be explored. In this study, small RNA and degradome libraries from peanut early pod of different developmental stages were constructed and sequenced. A total of 70 known and 24 novel miRNA families were discovered. Among them, 16 miRNA families were legume-specific and 12 families were peanut-specific. 30 known and 10 novel miRNA families were differentially expressed during pod development. In addition, 115 target genes were identified for 47 miRNA families by degradome sequencing. Several new targets that might be specific to peanut were found and further validated by RNA ligase-mediated rapid amplification of 5' cDNA ends (RLM 5'-RACE). Furthermore, we performed profiling analysis of intact and total transcripts of several target genes, demonstrating that SPL (miR156/157), NAC (miR164), PPRP (miR167 and miR1088), AP2 (miR172) and GRF (miR396) are actively modulated during early pod development, respectively. Large numbers of miRNAs and their related target genes were identified through deep sequencing. These findings provided new information on miRNA-mediated regulatory pathways in peanut pod, which will contribute to the comprehensive understanding of the molecular mechanisms that governing peanut embryo and early pod development.

Somatic Complaints in Early Adolescence: The Role of Parents' Emotion Socialization

ERIC Educational Resources Information Center

Kehoe, Christiane E.; Havighurst, Sophie S.; Harley, Ann E.

2015-01-01

This study investigated the relationship between parent emotion socialization and youth somatic complaints (SC) in an early adolescent sample using a longitudinal experimental design. An emotion-focused parenting intervention, which taught parent's skills to improve their emotional competence and emotion socialization, was used to examine whether…

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

PubMed Central

Rohrschneider, Monica R.; Nance, Jeremy

2013-01-01

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

Efficient embryonic culture method for the Japanese striped snake, Elaphe quadrivirgata, and its early developmental stages.

PubMed

Matsubara, Yoshiyuki; Sakai, Atsushi; Kuroiwa, Atsushi; Suzuki, Takayuki

2014-10-01

The morphogenesis of snake embryos is an elusive yet fascinating research target for developmental biologists. However, few data exist on development of early snake embryo due to limited availability of pregnant snakes, and the need to harvest early stage embryos directly from pregnant snakes before oviposition without knowing the date of fertilization. We established an ex vivo culture method for early snake embryos using the Japanese striped snake, Elaphe quadrivirgata. This method, which we named "sausage-style (SS) culture", allows us to harvest snake embryos at specific stages for each experiment. Using this SS culture system, we calculated somite formation rate at early stages before oviposition. The average somite formation rate between 6/7 and 12/13 somite stages was 145.9 min, between 60/70 and 80/91 somite stages 42.4 min, and between 113-115 and 126/127 somite stages 71 min. Thus, somite formation rate that we observed during early snake embryogenesis was changed over time. We also describe a developmental staging series for E. quadrivirgata. This is the first report of a developmental series of early snake embryogenesis prior to oviposition by full-color images with high-resolution. We propose that the SS culture system is an easy method for treating early snake embryos ex vivo. © 2014 The Authors Development, Growth & Differentiation © 2014 Japanese Society of Developmental Biologists.

Micropropagation of Crataeva adansonii D.C. Prodr: an ornamental avenue tree.

PubMed

Tyagi, Purnima; Sharma, P K; Kothari, S L

2010-01-01

In this chapter, we describe multiplication of the superior and elite tree of Crataeva adansonii using plant tissue culture techniques. An ornamental and avenue tree, it is not available in abundance because of poor seed germination and seedling establishment. It reproduces in nature by root suckers, but that restricts its distribution to very limited areas. Efficient procedures are outlined for plant regeneration through direct shoot bud formation, indirect organogenesis, and somatic embryogenesis through callus formation. Different explants were utilized for separate pathways of regeneration. Murashige and Skoog's (MS) medium containing 3 mg/L BA and 0.05-0.1 mg/L NAA is most effective in direct induction of axillary buds from nodal explants and shoot tips. Adventitious shoots developed from leaves on MS medium containing 3 mg/L BA and 0.1 mg/L NAA. De novo shoots were obtained from the anthers on MS medium supplemented with 3 mg/L BA. Somatic embryos developed on half strength MS medium containing 0.1 mg/L 2, 4-D. Roots were induced at the cut ends of shoots on MS basal medium devoid of growth regulators. The plantlets were then transferred to pots.

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.

Expression of a gymnosperm PIN homologous gene correlates with auxin immunolocalization pattern at cotyledon formation and in demarcation of the procambium during Picea abies somatic embryo development and in seedling tissues.

PubMed

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

2010-04-01

In seed plants, the body organization is established during embryogenesis and is uniform across gymnosperms and angiosperms, despite differences during early embryogeny. Evidence from angiosperms implicates the plant hormone auxin and its polar transport, mainly established by the PIN family of auxin efflux transporters, in the patterning of embryos. Here, PaPIN1 from Norway spruce (Picea abies [L.] Karst.), a gene widely expressed in conifer tissues and organs, was characterized and its expression and localization patterns were determined with reverse transcription polymerase chain reaction and in situ hybridization during somatic embryo development and in seedlings. PaPIN1 shares the predicted structure of other PIN proteins, but its central hydrophilic loop is longer than most PINs. In phylogenetic analyses, PaPIN1 clusters with Arabidopsis thaliana (L.) Heynh. PIN3, PIN4 and PIN7, but its expression pattern also suggests similarity to PIN1. The PaPIN1 expression signal was high in the protoderm of pre-cotyledonary embryos, but not if embryos were pre-treated with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). This, together with a high auxin immunolocalization signal in this cell layer, suggests a role of PaPIN1 during cotyledon formation. At later stages, high PaPIN1 expression was observed in differentiating procambium, running from the tip of incipient cotyledons down through the embryo axis and to the root apical meristem (RAM), although the mode of RAM specification in conifer embryos differs from that of most angiosperms. Also, the PaPIN1 in situ signal was high in seedling root tips including root cap columella cells. The results thus suggest that PaPIN1 provides an ancient function associated with auxin transport and embryo pattern formation prior to the separation of angiosperms and gymnosperms, in spite of some morphological differences.

In Silico Analysis of Small RNAs Suggest Roles for Novel and Conserved miRNAs in the Formation of Epigenetic Memory in Somatic Embryos of Norway Spruce.

PubMed

Yakovlev, Igor A; Fossdal, Carl G

2017-01-01

Epigenetic memory in Norway spruce affects the timing of bud burst and bud set, vitally important adaptive traits for this long-lived forest species. Epigenetic memory is established in response to the temperature conditions during embryogenesis. Somatic embryogenesis at different epitype inducing (EpI) temperatures closely mimics the natural processes of epigenetic memory formation in seeds, giving rise to epigenetically different clonal plants in a reproducible and predictable manner, with respect to altered bud phenology. MicroRNAs (miRNAs) and other small non-coding RNAs (sRNAs) play an essential role in the regulation of plant gene expression and may affect this epigenetic mechanism. We used NGS sequencing and computational in silico methods to identify and profile conserved and novel miRNAs among small RNAs in embryogenic tissues of Norway spruce at three EpI temperatures (18, 23 and 28°C). We detected three predominant classes of sRNAs related to a length of 24 nt, followed by a 21-22 nt class and a third 31 nt class of sRNAs. More than 2100 different miRNAs within the prevailing length 21-22 nt were identified. Profiling these putative miRNAs allowed identification of 1053 highly expressed miRNAs, including 523 conserved and 530 novels. 654 of these miRNAs were found to be differentially expressed (DEM) depending on EpI temperature. For most DEMs, we defined their putative mRNA targets. The targets represented mostly by transcripts of multiple-repeats proteins, like TIR, NBS-LRR, PPR and TPR repeat, Clathrin/VPS proteins, Myb-like, AP2, etc. Notably, 124 DE miRNAs targeted 203 differentially expressed epigenetic regulators. Developing Norway spruce embryos possess a more complex sRNA structure than that reported for somatic tissues. A variety of the predicted miRNAs showed distinct EpI temperature dependent expression patterns. These putative EpI miRNAs target spruce genes with a wide range of functions, including genes known to be involved in epigenetic regulation, which in turn could provide a feedback process leading to the formation of epigenetic marks. We suggest that TIR, NBS and LRR domain containing proteins could fulfill more general functions for signal transduction from external environmental stimuli and conversion them into molecular response. Fine-tuning of the miRNA production likely participates in both developmental regulation and epigenetic memory formation in Norway spruce.

Three-dimensional microCT imaging of murine embryonic development from immediate post-implantation to organogenesis: application for phenotyping analysis of early embryonic lethality in mutant animals.

PubMed

Ermakova, Olga; Orsini, Tiziana; Gambadoro, Alessia; Chiani, Francesco; Tocchini-Valentini, Glauco P

2018-04-01

In this work, we applied three-dimensional microCT imaging to study murine embryogenesis in the range from immediate post-implantation period (embryonic day 5.5) to mid-gestation (embryonic day 12.5) with the resolution up to 1.4 µm/voxel. Also, we introduce an imaging procedure for non-invasive volumetric estimation of an entire litter of embryos within the maternal uterine structures. This method allows for an accurate, detailed and systematic morphometric analysis of both embryonic and extra-embryonic components during embryogenesis. Three-dimensional imaging of unperturbed embryos was performed to visualize the egg cylinder, primitive streak, gastrulation and early organogenesis stages of murine development in the C57Bl6/N mouse reference strain. Further, we applied our microCT imaging protocol to determine the earliest point when embryonic development is arrested in a mouse line with knockout for tRNA splicing endonuclease subunit Tsen54 gene. Our analysis determined that the embryonic development in Tsen54 null embryos does not proceed beyond implantation. We demonstrated that application of microCT imaging to entire litter of non-perturbed embryos greatly facilitate studies to unravel gene function during early embryogenesis and to determine the precise point at which embryonic development is arrested in mutant animals. The described method is inexpensive, does not require lengthy embryos dissection and can be applicable for detailed analysis of mutant mice at laboratory scale as well as for high-throughput projects.

Tissue distribution and early developmental expression patterns of aldolase A, B, and C in grass carp Ctenopharyngodon idellus.

PubMed

Fan, J J; Bai, J J; Ma, D M; Yu, L Y; Jiang, P

2017-09-27

Aldolase is a key enzyme involved in glycolysis, gluconeogenesis, and the pentose phosphate pathway. To establish the expression patterns of all three aldolase isozyme genes in different tissues and during early embryogenesis in lower vertebrates, as well as to explore the functional differences between these three isozymes, the grass carp was selected as a model owing to its relatively high glucose-metabolizing capability. Based on the cDNA sequences of the aldolase A, B, and C genes, the expression patterns of these three isozymes were analyzed in different tissues and during early embryogenesis using quantitative real-time polymerase chain reaction (qRT-PCR). Sequence analysis of cDNAs indicated that aldolase A, B, and C (GenBank accession numbers: KM192250, KM192251, and KM192252) consist of 364, 364, and 363 amino acids, respectively. The qRT-PCR results showed that the expression levels of aldolase A, B, and C were highest in the muscle, liver, and brain, respectively. Aldolase A and C exhibited similar expression patterns during embryogenesis, with high levels observed in unfertilized and fertilized eggs and at the blastocyst stage, followed by a decline and then increase after organogenesis. In contrast, aldolase B transcript was not detected during the unfertilized egg stage, and appeared only from gastrulation; the expression increased markedly during the feeding period (72 h after hatching), at which point the level was higher than those of aldolase A and C. These data suggest that the glucose content of grass carp starter feed should be adjusted according to the metabolic activity of aldolase B.

NEUROENDOCRINE AND REPRODUCTIVE EFFECTS OF PESTICIDES

EPA Science Inventory

Neuroendocrine and Reproductive Effects of Pesticides

1Stoker, TE, Goldman 2, JM and Cooper 2, RL.

1Gamete and Early Embryogenesis Biology Branch and 2 Endocrinology Branch, Reproductive Toxicology Division, National Health and Environmental Effects Research Laborat...

De novo DNA methylation during monkey pre-implantation embryogenesis.

PubMed

Gao, Fei; Niu, Yuyu; Sun, Yi Eve; Lu, Hanlin; Chen, Yongchang; Li, Siguang; Kang, Yu; Luo, Yuping; Si, Chenyang; Yu, Juehua; Li, Chang; Sun, Nianqin; Si, Wei; Wang, Hong; Ji, Weizhi; Tan, Tao

2017-04-01

Critical epigenetic regulation of primate embryogenesis entails DNA methylome changes. Here we report genome-wide composition, patterning, and stage-specific dynamics of DNA methylation in pre-implantation rhesus monkey embryos as well as male and female gametes studied using an optimized tagmentation-based whole-genome bisulfite sequencing method. We show that upon fertilization, both paternal and maternal genomes undergo active DNA demethylation, and genome-wide de novo DNA methylation is also initiated in the same period. By the 8-cell stage, remethylation becomes more pronounced than demethylation, resulting in an increase in global DNA methylation. Promoters of genes associated with oxidative phosphorylation are preferentially remethylated at the 8-cell stage, suggesting that this mode of energy metabolism may not be favored. Unlike in rodents, X chromosome inactivation is not observed during monkey pre-implantation development. Our study provides the first comprehensive illustration of the 'wax and wane' phases of DNA methylation dynamics. Most importantly, our DNA methyltransferase loss-of-function analysis indicates that DNA methylation influences early monkey embryogenesis.

De novo DNA methylation during monkey pre-implantation embryogenesis

PubMed Central

Gao, Fei; Niu, Yuyu; Sun, Yi Eve; Lu, Hanlin; Chen, Yongchang; Li, Siguang; Kang, Yu; Luo, Yuping; Si, Chenyang; Yu, Juehua; Li, Chang; Sun, Nianqin; Si, Wei; Wang, Hong; Ji, Weizhi; Tan, Tao

2017-01-01

Critical epigenetic regulation of primate embryogenesis entails DNA methylome changes. Here we report genome-wide composition, patterning, and stage-specific dynamics of DNA methylation in pre-implantation rhesus monkey embryos as well as male and female gametes studied using an optimized tagmentation-based whole-genome bisulfite sequencing method. We show that upon fertilization, both paternal and maternal genomes undergo active DNA demethylation, and genome-wide de novo DNA methylation is also initiated in the same period. By the 8-cell stage, remethylation becomes more pronounced than demethylation, resulting in an increase in global DNA methylation. Promoters of genes associated with oxidative phosphorylation are preferentially remethylated at the 8-cell stage, suggesting that this mode of energy metabolism may not be favored. Unlike in rodents, X chromosome inactivation is not observed during monkey pre-implantation development. Our study provides the first comprehensive illustration of the 'wax and wane' phases of DNA methylation dynamics. Most importantly, our DNA methyltransferase loss-of-function analysis indicates that DNA methylation influences early monkey embryogenesis. PMID:28233770

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.

Morphogenesis in leaf and single-cell cultures of mature Juniperus oxycedrus.

PubMed

Gomez, M P; Segura, J

1996-08-01

Single cells were mechanically isolated from leaf-derived callus of mature Juniperus oxycedrus L. These cells divided and gave rise to callus when plated on medium containing growth regulators. Best plating efficiency was obtained on a modified Schenk and Hildebrandt medium supplemented with 0.6 micro M 2,4-dichlorophenoxyacetic acid and 100 mg l(-1) casein hydrolyzate. Although single-cell-derived callus showed poor morphogenic potential, both adventitious shoots and embryogenic tissues differentiated from the callus. We also achieved induction of somatic embryogenesis in leaf explants of mature J. oxycedrus trees cultured in the presence of 6.0 or 10.0 micro M 2,4-dichlorophenoxyacetic acid or picloram. Frequency of embryogenic callus ranged from 6 to 18%; however, under the culture conditions tested, isolated embryos failed to develop into plants.

Improving Cotton Embryo Culture by Simulating In Ovulo Nutrient and Hormone Levels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodney Fuller; Vincent Liddiard; J. Hess

Plant ovules provide zygotes with a physicochemical environment that supports embryo differentiation, growth, and maturation. The exact nature of this embryogenesis-enabling environment is not well characterized, as evidenced by failed attempts to induce normal embryony from zygotes or proembryos (precotyledonary) on defined media. To identify factors required for cotton (Gossypium hirsutum L.) zygotic embryony in vitro, we previously performed chemical and dissolved oxygen tension analyses of cotton ovule fluids and tissues at multiple stages of embryony in situ. Based on these analyses, we report herein the development of procedures that normalize embryo differentiation, growth, maturation, and germination in vitro, startingmore » with proembryos. Our medium differed from Murashige and Skoog (MS) medium as follows (percentage of MS): N (30%, mostly from ten amino acids), P (815%), K (237%), Mg (85%), Ca (267%), S (506%), Fe (88%), and myoinositol (883%). Levels of other MS nutrients and vitamins, except sucrose, were kept at MS levels. Additionally, we included 100 mg L-1 casein hydrolysate plus the following (mmol L-1): d-glucose (1.8), fructose (4.7), sucrose (62.0), arabinose (7.1), melibiose (3.5), malic acid (11.6), and citric acid (3.8). Mannitol was added to achieve a medium osmotic potential of -1.10 MPa, and an atmospheric O2 tension of 3.3 mol m-3 at the surface of embryos was maintained during culture. When cultured on medium containing 8.0 µmol L-1 indole-3-acetic acid, 80-90% of proembryos (as small as 100 cells) of cultivars HS-26 and B-27 increased four- to eightfold in surface area during the first 18 d in culture and germinated thereafter to produce viable plants. Increases in surface area of proembryos cultured on a modified MS medium previously used for somatic embryogenesis were from 0.2- to 0.6-fold. The described embryo culture medium should be useful for studying nutritional and molecular aspects of early embryony and possibly for plant zygote transformation protocols.« less

Transcriptional Ontogeny of the Developing Liver

EPA Science Inventory

During embryogenesis the liver is derived from endodermal cells lining the digestive tract. These endodermal progenitor cells contribute to forming the parenchyma of a number of organs including the liver and pancreas. Early in organogenesis the fetal liver is populated by hemato...

Interspecies chimeras.

PubMed

Suchy, Fabian; Nakauchi, Hiromitsu

2018-05-30

By probing early embryogenesis and regeneration, interspecies chimeras provide a unique platform for discovery and clinical use. Although efficient generation of human:animal chimeric embryos remains elusive, recent advancements attempt to overcome incompatibilities in xenogeneic development and transplantation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Conserved elements in the nanos3 3'UTR of olive flounder are responsible for the selective retention of RNA in germ cells.

PubMed

Li, Meijie; Tan, Xungang; Sui, Yulei; Jiao, Shuang; Wu, Zhihao; You, Feng

2016-08-01

In teleost fish, primordial germ cells (PGCs) are specified very early during embryogenesis and migrate to the site that gonads are formed. A previous study indicated that nanos3 is specifically expressed in PGCs, and the 3' untranslated region (UTR) of nanos3 is responsible for the localization of mRNA in these cells. In this study, we aimed to investigate the functional regions of nanos3 3'UTR in olive flounder using truncated and mutated nanos3 3'UTRs fused to chimeric RNAs and microinjected into fertilized zebrafish eggs. The results indicated that a 68-bp functional element in the nanos3 3'UTR of olive flounder played important roles in the protection and degradation of RNA. Within this element, a U-rich region was identified to be responsible for the protection of RNA in PGCs and two GCAC sites for the degradation of RNA in somatic cells. The first GCAC was located adjacently to the U-rich region and the second GCAC within the U-rich region. Overall, we concluded that the two GCACs were the binding sites of miR-430, a microRNA that suppresses translation, whereas the U-rich region was the binding site of Dnd, a protein that antagonizes the miR-430-mediated silencing of mRNA. Copyright © 2016 Elsevier Inc. All rights reserved.

Mated Progeny Production Is a Biomarker of Aging in Caenorhabditis elegans

PubMed Central

Pickett, Christopher L.; Dietrich, Nicholas; Chen, Junfang; Xiong, Chengjie; Kornfeld, Kerry

2013-01-01

The relationships between reproduction and aging are important for understanding the mechanisms of aging and evaluating evolutionary theories of aging. To investigate the effects of progeny production on reproductive and somatic aging, we conducted longitudinal studies of Caenorhabditis elegans hermaphrodites. For mated wild-type animals that were not sperm limited and survived past the end of the reproductive period, high levels of cross-progeny production were positively correlated with delayed reproductive and somatic aging. In this group of animals, individuals that generated more cross progeny also reproduced and lived longer than individuals that generated fewer cross progeny. These results indicate that progeny production does not accelerate reproductive or somatic aging. This longitudinal study demonstrated that cumulative cross progeny production through day four is an early-stage biomarker that is a positive predictor of longevity. Furthermore, in mated animals, high levels of early cross progeny production were positively correlated with high levels of late cross progeny production, indicating that early progeny production does not accelerate reproductive aging. The relationships between progeny production and aging were further evaluated by comparing self-fertile hermaphrodites that generated relatively few self progeny with mated hermaphrodites that generated many cross progeny. The timing of age-related somatic degeneration was similar in these groups, suggesting progeny production does not accelerate somatic aging. These studies rigorously define relationships between progeny production, reproductive aging, and somatic aging and identify new biomarkers of C. elegans aging. These results indicate that some mechanisms or pathways control age-related degeneration of both reproductive and somatic tissues in C. elegans. PMID:24142929

Overexpression of VpPR10.1 by an efficient transformation method enhances downy mildew resistance in V. vinifera.

PubMed

Su, Hang; Jiao, Yun-Tong; Wang, Fang-Fang; Liu, Yue-E; Niu, Wei-Li; Liu, Guo-Tian; Xu, Yan

2018-05-01

Putrescine and spermidine increase the transformation efficiency of Vitis vinifera L. cv. Thompson seedless. Accumulation of VpPR10.1 in transgenic V. vinifera Thompson seedless, likely increases its resistance to downy mildew. A more efficient method is described for facilitating Agrobacterium-mediated transformation of Vitis vinifera L. cv. Thompson Seedless somatic embryogenesis using polyamines (PAs). The efficacies of putrescine, spermidine and spermine are identified at a range of concentrations (10 µM, 100 µM and 1 mM) added to the culture medium during somatic embryo growth. Putrescine (PUT) and spermidine (SPD) promote the recovery of proembryonic masses (PEM) and the development of somatic embryos (SE) after co-cultivation. Judging from the importance of the time-frame in genetic transformation, PAs added at the co-cultivation stage have a stronger effect than delayed selection treatments, which are superior to antibiotic treatments in the selection stage. Best embryogenic responses are with 1 mM PUT and 100 µM SPD added to the co-culture medium. Using the above method, a pathogenesis-related gene (VpPR10.1) from Chinese wild Vitis pseudoreticulata was transferred into Thompson Seedless for functional evaluation. The transgenic line, confirmed by western blot analysis, was inoculated with Plasmopara viticola to test for downy mildew resistance. Based on observed restrictions of hyphal growth and increases in H 2 O 2 accumulation in the transgenic plants, the accumulation of VpPR10.1 likely enhanced the transgenic plants resistance to downy mildew.

Cloning and expression of 1-aminocyclopropane-1-carboxylate oxidase cDNA induced by thidiazuron during somatic embryogenesis of alfalfa (Medicago sativa).

PubMed

Feng, Bi-Hong; Wu, Bei; Zhang, Chun-Rong; Huang, Xia; Chen, Yun-Feng; Huang, Xue-Lin

2012-01-15

Embryogenic callus (EC) induced from petioles of alfalfa (Medicago sativa L. cv. Jinnan) on B5h medium turned green, compact and non-embryogenic when the kinetin (KN) in the medium was replaced partially or completely by thidiazuron (TDZ). The application of CoCl₂, which is an inhibitor of 1-aminocyclopropane-1-carboxylate oxidase (ACO), counteracted the effect of TDZ. Ethylene has been shown to be involved in the modulation of TDZ-induced morphogenesis responses. However, very little is known about the genes involved in ethylene formation during somatic embryogenesis (SE). To investigate whether ethylene mediated by ACO is involved in the effect of TDZ on inhibition of embryogenic competence of the alfalfa callus. In this study we cloned full-length ACO cDNA from the alfalfa callus, named MsACO, and observed changes in this gene expression during callus formation and induction of SE under treatment with TDZ or TDZ plus CoCl₂. RNA blot analysis showed that during the EC subcultural period, the expression level of MsACO in EC was significantly increased on the 2nd day, rose to the highest level on the 8th day and remained at this high level until the 21st day. However, the ACO expression in the TDZ (0.93 μM)-treated callus was higher than in the EC especially on the 8th day. Moreover the ACO expression level increased with increasing TDZ concentration during the subcultural/maintenance period of the callus. It is worth noting that comparing the treatment with TDZ alone, the treatment with 0.93 μM TDZ plus 50 μM CoCl₂ reduced both of the ACO gene expressions and ACO activity in the treated callus. These results indicate that the effect of TDZ could be counteracted by CoCl₂ either on the ACO gene expression level or ACO activity. Thus, a TDZ inhibitory effect on embryogenic competence of alfalfa callus could be mediated by ACO gene expression. Crown Copyright © 2011. Published by Elsevier GmbH. All rights reserved.

Self-organization of human iPS cells into trophectoderm mimicking cysts induced by adhesion restriction using microstructured mesh scaffolds.

PubMed

Okeyo, Kennedy O; Tanabe, Maiko; Kurosawa, Osamu; Oana, Hidehiro; Washizu, Masao

2018-04-01

Cellular dynamics leading to the formation of the trophectoderm in humans remain poorly understood owing to limited accessibility to human embryos for research into early human embryogenesis. Compared to animal models, organoids formed by self-organization of stem cells in vitro may provide better insights into differentiation and complex morphogenetic processes occurring during early human embryogenesis. Here we demonstrate that modulating the cell culture microenvironment alone can trigger self-organization of human induced pluripotent stem cells (hiPSCs) to yield trophectoderm-mimicking cysts without chemical induction. To modulate the adhesion microenvironment, we used the mesh culture technique recently developed by our group, which involves culturing hiPSCs on suspended micro-structured meshes with limited surface area for cell adhesion. We show that this adhesion-restriction strategy can trigger a two-stage self-organization of hiPSCs; first into stem cell sheets, which express pluripotency signatures until around day 8-10, then into spherical cysts following differentiation and self-organization of the sheet-forming cells. Detailed morphological analysis using immunofluorescence microscopy with both confocal and two-photon microscopes revealed the anatomy of the cysts as consisting of a squamous epithelial wall richly expressing E-cadherin and CDX2. We also confirmed that the cysts exhibit a polarized morphology with basal protrusions, which show migratory behavior when anchored. Together, our results point to the formation of cysts which morphologically resemble the trophectoderm at the late-stage blastocyst. Thus, the mesh culture microenvironment can initiate self-organization of hiPSCs into trophectoderm-mimicking cysts as organoids with potential application in the study of early embryogenesis and also in drug development. © 2018 Japanese Society of Developmental Biologists.

Pattern formation in early embryogenesis of Xenopus laevis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mglinets, V.A.

1995-07-01

Establishment of egg polarity, separation of germ layers, and the appearance of animal-vegetal, dorsoventral, and anteroposterior axes in Xenopus laevis embryos are considered. The control of these processes by gene coding for growth factors, protooncogens, and homeobox-containing genes is also been reviewed.

FLASH is essential during early embryogenesis and cooperates with p73 to regulate histone gene transcription.

PubMed

De Cola, A; Bongiorno-Borbone, L; Bianchi, E; Barcaroli, D; Carletti, E; Knight, R A; Di Ilio, C; Melino, G; Sette, C; De Laurenzi, V

2012-02-02

Replication-dependent histone gene expression is a fundamental process occurring in S-phase under the control of the cyclin-E/CDK2 complex. This process is regulated by a number of proteins, including Flice-Associated Huge Protein (FLASH) (CASP8AP2), concentrated in specific nuclear organelles known as HLBs. FLASH regulates both histone gene transcription and mRNA maturation, and its downregulation in vitro results in the depletion of the histone pull and cell-cycle arrest in S-phase. Here we show that the transcription factor p73 binds to FLASH and is part of the complex that regulates histone gene transcription. Moreover, we created a novel gene trap to disrupt FLASH in mice, and we show that homozygous deletion of FLASH results in early embryonic lethality, owing to arrest of FLASH(-/-) embryos at the morula stage. These results indicate that FLASH is an essential, non-redundant regulator of histone transcription and cell cycle during embryogenesis.

A cell-based computational model of early embryogenesis coupling mechanical behaviour and gene regulation

NASA Astrophysics Data System (ADS)

Delile, Julien; Herrmann, Matthieu; Peyriéras, Nadine; Doursat, René

2017-01-01

The study of multicellular development is grounded in two complementary domains: cell biomechanics, which examines how physical forces shape the embryo, and genetic regulation and molecular signalling, which concern how cells determine their states and behaviours. Integrating both sides into a unified framework is crucial to fully understand the self-organized dynamics of morphogenesis. Here we introduce MecaGen, an integrative modelling platform enabling the hypothesis-driven simulation of these dual processes via the coupling between mechanical and chemical variables. Our approach relies upon a minimal `cell behaviour ontology' comprising mesenchymal and epithelial cells and their associated behaviours. MecaGen enables the specification and control of complex collective movements in 3D space through a biologically relevant gene regulatory network and parameter space exploration. Three case studies investigating pattern formation, epithelial differentiation and tissue tectonics in zebrafish early embryogenesis, the latter with quantitative comparison to live imaging data, demonstrate the validity and usefulness of our framework.

Microgravity effects of sea urchin fertilization and development

NASA Technical Reports Server (NTRS)

Steffen, S.; Simerly, C.; Schatten, H.; Schatten, G.; Fiser, R.

1992-01-01

Gravity has been a pervasive influence on all living systems and there is convincing evidence to suggest that it alters fertilization and embryogenesis in several developmental systems. Notwithstanding the global importance of gravity on development, it has only been recently possible to begin to design experiments which might directly investigate the specific effects of this vector. The goal of this research program is to explore and understand the effects of gravity on fertilization and early development using sea urchins as a model system. Sea urchin development has several advantages for this project including the feasibility of maintaining and manipulating these cells during spaceflight, the high percentage of normal fertilization and early development, and the abundant knowledge about molecular, biochemical, and cellular events during embryogenesis which permits detailed insights into the mechanism by which gravity might interfere with development. Furthermore, skeletal calcium is deposited into the embryonic spicules within a day of fertilization permitting studies of the effects of gravity on bone calcium deposition.

Embryological origin of the endocardium and derived valve progenitor cells: from developmental biology to stem cell-based valve repair.

PubMed

Pucéat, Michel

2013-04-01

The cardiac valves are targets of both congenital and acquired diseases. The formation of valves during embryogenesis (i.e., valvulogenesis) originates from endocardial cells lining the myocardium. These cells undergo an endothelial-mesenchymal transition, proliferate and migrate within an extracellular matrix. This leads to the formation of bilateral cardiac cushions in both the atrioventricular canal and the outflow tract. The embryonic origin of both the endocardium and prospective valve cells is still elusive. Endocardial and myocardial lineages are segregated early during embryogenesis and such a cell fate decision can be recapitulated in vitro by embryonic stem cells (ESC). Besides genetically modified mice and ex vivo heart explants, ESCs provide a cellular model to study the early steps of valve development and might constitute a human therapeutic cell source for decellularized tissue-engineered valves. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction. Copyright © 2012 Elsevier B.V. All rights reserved.

Aberrant ligand-induced activation of G protein-coupled estrogen receptor 1 (GPER) results in developmental malformations during vertebrate embryogenesis.

PubMed

Jayasinghe, B Sumith; Volz, David C

2012-01-01

G protein-coupled estrogen receptor 1 (GPER) is a G protein-coupled receptor (GPCR) unrelated to nuclear estrogen receptors but strongly activated by 17β-estradiol in both mammals and fish. To date, the distribution and functional characterization of GPER within reproductive and nonreproductive vertebrate organs have been restricted to juvenile and adult animals. In contrast, virtually nothing is known about the spatiotemporal distribution and function of GPER during vertebrate embryogenesis. Using zebrafish as an animal model, we investigated the potential functional role and expression of GPER during embryogenesis. Based on real-time PCR and whole-mount in situ hybridization, gper was expressed as early as 1 h postfertilization (hpf) and exhibited strong stage-dependent expression patterns during embryogenesis. At 26 and 38 hpf, gper mRNA was broadly distributed throughout the body, whereas from 50 to 98 hpf, gper expression was increasingly localized to the heart, brain, neuromasts, craniofacial region, and somite boundaries of developing zebrafish. Continuous exposure to a selective GPER agonist (G-1)-but not continuous exposure to a selective GPER antagonist (G-15)-from 5 to 96 hpf, or within three developmental windows ranging from 10 to 72 hpf, resulted in adverse concentration-dependent effects on survival, gross morphology, and somite formation within the trunk of developing zebrafish embryos. Importantly, based on co-exposure studies, G-15 blocked severe G-1-induced developmental toxicity, suggesting that G-1 toxicity is mediated via aberrant activation of GPER. Overall, our findings suggest that xenobiotic-induced GPER activation represents a potentially novel and understudied mechanism of toxicity for environmentally relevant chemicals that affect vertebrate embryogenesis.

The Roles of the Wnt-Antagonists Axin and Lrp4 during Embryogenesis of the Red Flour Beetle Tribolium castaneum

PubMed Central

Prühs, Romy

2017-01-01

In both vertebrates and invertebrates, the Wnt-signaling pathway is essential for numerous processes in embryogenesis and during adult life. Wnt activity is fine-tuned at various levels by the interplay of a number of Wnt-agonists (Wnt ligands, Frizzled-receptors, Lrp5/6 coreceptors) and Wnt-antagonists (among them Axin, Secreted frizzled and Lrp4) to define anterior–posterior polarity of the early embryo and specify cell fate in organogenesis. So far, the functional analysis of Wnt-pathway components in insects has concentrated on the roles of Wnt-agonists and on the Wnt-antagonist Axin. We depict here additional features of the Wnt-antagonist Axin in the flour beetle Tribolium castaneum. We show that Tc-axin is dynamically expressed throughout embryogenesis and confirm its essential role in head development. In addition, we describe an as yet undetected, more extreme Tc-axin RNAi-phenotype, the ectopic formation of posterior abdominal segments in reverse polarity and a second hindgut at the anterior. For the first time, we describe here that an lrp4 ortholog is involved in axis formation in an insect. The Tribolium Lrp4 ortholog is ubiquitously expressed throughout embryogenesis. Its downregulation via maternal RNAi results in the reduction of head structures but not in axis polarity reversal. Furthermore, segmentation is impaired and larvae develop with a severe gap-phenotype. We conclude that, as in vertebrates, Tc-lrp4 functions as a Wnt-inhibitor in Tribolium during various stages of embryogenesis. We discuss the role of both components as negative modulators of Wnt signaling in respect to axis formation and segmentation in Tribolium. PMID:29615567

Genetic diversity and geographical dispersal in grapevine clones revealed by microsatellite markers.

PubMed

Moncada, Ximena; Pelsy, Frédérique; Merdinoglu, Didier; Hinrichsen, Patricio

2006-11-01

Intravarietal genetic diversification associated with geographical dispersal of a vegetatively propagated species was studied using grapevine Vitis vinifera L. 'Cabernet Sauvignon' as a model. Fifty-nine clonal samples obtained from 7 countries (France, Chile, Spain, Australia, Hungary, USA, and Italy) were analyzed using 84 microsatellite markers. Eighteen polymorphic microsatellite loci (21.4%) were detected, finding 22 different genotypes in the population analyzed with a genetic similarity of over 97%. The presence of chimeric clones was evidenced at locus VMC5g7 by means of a segregation analysis of descendants by self-pollination of a triallelic Chilean clone and by somatic embryogenesis analysis, showing a mutation in L2 cell layer. Only 2 clones (obtained from France and Australia) presented the ancestral genotype, and the most divergent genotype was exhibited by another French clone, which had accumulated 5 somatic mutations. The 2 largest populations considered (from France and Chile) showed a clear divergency in the polymorphisms detected. These antecedents enabled the tracing of geographical dispersal with a phylogenetic hypothesis supporting France as the center of origin of diversification of Cabernet Sauvignon. The results obtained could help to explain diversification processes in other grapevine cultivars. The possibility that this kind of genetic variability occurs in other vegetatively propagated species is discussed, focusing on possible fingerprinting applications.

Translatome analysis at the egg-to-embryo transition in sea urchin

PubMed Central

Chassé, Héloïse; Aubert, Julie; Boulben, Sandrine; Le Corguillé, Gildas; Corre, Erwan; Cormier, Patrick

2018-01-01

Abstract Early embryogenesis relies on the translational regulation of maternally stored mRNAs. In sea urchin, fertilization triggers a dramatic rise in translation activity, necessary for the onset of cell division. Here, the full spectrum of the mRNAs translated upon fertilization was investigated by polysome profiling and sequencing. The translatome of the early sea urchin embryo gave a complete picture of the polysomal recruitment dynamics following fertilization. Our results indicate that only a subset of maternal mRNAs were selectively recruited onto polysomes, with over-represented functional categories in the translated set. The increase in translation upon fertilization depends on the formation of translation initiation complexes following mTOR pathway activation. Surprisingly, mTOR pathway inhibition differentially affected polysomal recruitment of the newly translated mRNAs, which thus appeared either mTOR-dependent or mTOR-independent. Therefore, our data argue for an alternative to the classical cap-dependent model of translation in early development. The identification of the mRNAs translated following fertilization helped assign translational activation events to specific mRNAs. This translatome is the first step to a comprehensive analysis of the molecular mechanisms governing translation upon fertilization and the translational regulatory networks that control the egg-to-embryo transition as well as the early steps of embryogenesis. PMID:29660001

Translatome analysis at the egg-to-embryo transition in sea urchin.

PubMed

Chassé, Héloïse; Aubert, Julie; Boulben, Sandrine; Le Corguillé, Gildas; Corre, Erwan; Cormier, Patrick; Morales, Julia

2018-05-18

Early embryogenesis relies on the translational regulation of maternally stored mRNAs. In sea urchin, fertilization triggers a dramatic rise in translation activity, necessary for the onset of cell division. Here, the full spectrum of the mRNAs translated upon fertilization was investigated by polysome profiling and sequencing. The translatome of the early sea urchin embryo gave a complete picture of the polysomal recruitment dynamics following fertilization. Our results indicate that only a subset of maternal mRNAs were selectively recruited onto polysomes, with over-represented functional categories in the translated set. The increase in translation upon fertilization depends on the formation of translation initiation complexes following mTOR pathway activation. Surprisingly, mTOR pathway inhibition differentially affected polysomal recruitment of the newly translated mRNAs, which thus appeared either mTOR-dependent or mTOR-independent. Therefore, our data argue for an alternative to the classical cap-dependent model of translation in early development. The identification of the mRNAs translated following fertilization helped assign translational activation events to specific mRNAs. This translatome is the first step to a comprehensive analysis of the molecular mechanisms governing translation upon fertilization and the translational regulatory networks that control the egg-to-embryo transition as well as the early steps of embryogenesis.

DNA methylation dynamics during early plant life.

PubMed

Bouyer, Daniel; Kramdi, Amira; Kassam, Mohamed; Heese, Maren; Schnittger, Arp; Roudier, François; Colot, Vincent

2017-09-25

Cytosine methylation is crucial for gene regulation and silencing of transposable elements in mammals and plants. While this epigenetic mark is extensively reprogrammed in the germline and early embryos of mammals, the extent to which DNA methylation is reset between generations in plants remains largely unknown. Using Arabidopsis as a model, we uncovered distinct DNA methylation dynamics over transposable element sequences during the early stages of plant development. Specifically, transposable elements and their relics show invariably high methylation at CG sites but increasing methylation at CHG and CHH sites. This non-CG methylation culminates in mature embryos, where it reaches saturation for a large fraction of methylated CHH sites, compared to the typical 10-20% methylation level observed in seedlings or adult plants. Moreover, the increase in CHH methylation during embryogenesis matches the hypomethylated state in the early endosperm. Finally, we show that interfering with the embryo-to-seedling transition results in the persistence of high CHH methylation levels after germination, specifically over sequences that are targeted by the RNA-directed DNA methylation (RdDM) machinery. Our findings indicate the absence of extensive resetting of DNA methylation patterns during early plant life and point instead to an important role of RdDM in reinforcing DNA methylation of transposable element sequences in every cell of the mature embryo. Furthermore, we provide evidence that this elevated RdDM activity is a specific property of embryogenesis.

Dual specificity of activin type II receptor ActRIIb in dorso-ventral patterning during zebrafish embryogenesis.

PubMed

Nagaso, H; Suzuki, A; Tada, M; Ueno, N

1999-04-01

Members of the transforming growth factor-beta (TGF-beta) superfamily are thought to regulate specification of a variety of tissue types in early embryogenesis. These effects are mediated through a cell surface receptor complex, consisting of two classes of ser/thr kinase receptor, type I and type II. In the present study, cDNA encoding zebrafish activin type II receptors, ActRIIa and ActRIIb was cloned and characterized. Overexpression of ActRIIb in zebrafish embryos caused dorsalization of embryos, as observed in activin-overexpressing embryos. However, in blastula stage embryos, ActRIIb induced formation of both dorsal and ventro-lateral mesoderm. It has been suggested that these inducing signals from ActRIIb are mediated through each specific type I receptor, TARAM-A and BMPRIA, depending on activin and bone morphogenetic protein (BMP), respectively. In addition, it was shown that a kinase-deleted form of ActRIIb (dnActRIIb) suppressed both activin- and BMP-like signaling pathways. These results suggest that ActRIIb at least has dual roles in both activin and BMP signaling pathways during zebrafish embryogenesis.

Homologue of Sox10 in Misgurnus anguillicaudatus: sequence, expression pattern during early embryogenesis.

PubMed

Xia, Xiaohua; Nan, Ping; Zhang, Linxia; Sun, Jinsheng; Chang, Zhongjie

2013-10-01

A number of genetic studies have established that Sox10 is a transcription factor associated with neurogenesis in vertebrates. We have isolated a homologue of Sox10 gene from the brain of Misgurnus anguillicaudatus by using homologous cloning and RACE method, designated as MaSox10b. The full-length cDNA of MaSox10b contained a 311 bp 5'UTR, a 312 bp 3'UTR and an ORF encoding a putative protein of 490 amino acids with a characteristic HMG-box DNA-binding domain of 79 amino acids (aa: 105-183). Phylogenetic tree shows that the MaSOX10b fits within the Sox10 clade and clusters firmly into Sox10b branches. During embryogenesis, MaSox10b was first detected in gastrulae stage. From somitogenesis stage and thereafter, distinct expression was observed in the medial neural tube, extending from the hindbrain through the posterior trunk. Taken together, these preliminary findings suggested that MaSox10b is highly conserved during vertebrate evolution and involved in a wide range of developmental processes including embryogenesis and neurogenesis.

The impact of transposable elements on mammalian development

PubMed Central

Garcia-Perez, Jose L.; Widmann, Thomas J.; Adams, Ian R.

2018-01-01

Summary Despite often being classified as selfish or junk DNA, transposable elements (TEs) are a group of abundant genetic sequences that significantly impact on mammalian development and genome regulation. In recent years, our understanding of how pre-existing TEs affect genome architecture, gene regulatory networks and protein function during mammalian embryogenesis has dramatically expanded. In addition, the mobilization of active TEs in selected cell types has been shown to generate genetic variation during development and in fully differentiated tissues. Importantly, the ongoing domestication and evolution of TEs appears to provide a rich source of regulatory elements, functional modules and genetic variation that fuels the evolution of mammalian developmental processes. Here, we review the functional impact that TEs exert on mammalian developmental processes and how the somatic activity of TEs can influence gene regulatory networks. PMID:27875251

A look at plant immunity through the window of the multitasking coreceptor BAK1.

PubMed

Yasuda, Shigetaka; Okada, Kentaro; Saijo, Yusuke

2017-08-01

Recognition of microbe- and danger-associated molecular patterns (MAMPs and DAMPs, respectively) by pattern recognition receptors (PRRs) is central to innate immunity in both plants and animals. The plant PRRs described to date are all cell surface-localized receptors. According to their ligand-binding ectodomains, each PRR engages a specific coreceptor or adaptor kinase in its signaling complexes to regulate defense signaling. With a focus on the coreceptor RLK BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) and related SOMATIC EMBRYOGENESIS RECEPTOR KINASEs (SERKs), here we review the increasing inventory of BAK1 partners and their functions in plant immunity. We also discuss the significance of autoimmunity triggered by BAK1/SERK4 disintegration in shaping the strategies for attenuation of PRR signaling by infectious microbes and host plants. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structure of the OsSERK2 leucine-rich repeat extracellular domain.

PubMed

McAndrew, Ryan; Pruitt, Rory N; Kamita, Shizuo G; Pereira, Jose Henrique; Majumdar, Dipali; Hammock, Bruce D; Adams, Paul D; Ronald, Pamela C

2014-11-01

Somatic embryogenesis receptor kinases (SERKs) are leucine-rich repeat (LRR)-containing integral membrane receptors that are involved in the regulation of development and immune responses in plants. It has recently been shown that rice SERK2 (OsSERK2) is essential for XA21-mediated resistance to the pathogen Xanthomonas oryzae pv. oryzae. OsSERK2 is also required for the BRI1-mediated, FLS2-mediated and EFR-mediated responses to brassinosteroids, flagellin and elongation factor Tu (EF-Tu), respectively. Here, crystal structures of the LRR domains of OsSERK2 and a D128N OsSERK2 mutant, expressed as hagfish variable lymphocyte receptor (VLR) fusions, are reported. These structures suggest that the aspartate mutation does not generate any significant conformational change in the protein, but instead leads to an altered interaction with partner receptors.

Reverse engineering the mechanical and molecular pathways in stem cell morphogenesis.

PubMed

Lu, Kai; Gordon, Richard; Cao, Tong

2015-03-01

The formation of relevant biological structures poses a challenge for regenerative medicine. During embryogenesis, embryonic cells differentiate into somatic tissues and undergo morphogenesis to produce three-dimensional organs. Using stem cells, we can recapitulate this process and create biological constructs for therapeutic transplantation. However, imperfect imitation of nature sometimes results in in vitro artifacts that fail to recapitulate the function of native organs. It has been hypothesized that developing cells may self-organize into tissue-specific structures given a correct in vitro environment. This proposition is supported by the generation of neo-organoids from stem cells. We suggest that morphogenesis may be reverse engineered to uncover its interacting mechanical pathway and molecular circuitry. By harnessing the latent architecture of stem cells, novel tissue-engineering strategies may be conceptualized for generating self-organizing transplants. Copyright © 2013 John Wiley & Sons, Ltd.

Role of Abcg2 During Mouse Embroyonic Stem Cell Diffferentiation

EPA Science Inventory

Role of Abcg2 During Mouse Embryonic Stem Cell Differentiation. Abcg2 is a multidrug resistance ATP-binding cassette (ABC) transporter whose activity may be considered a hallmark of stem cell plasticity. The role of Abcg2 during early embryogenesis, however, is unclear. Studies...

(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. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Crucial stages of embryogenesis of R. arvalis: Part 1. Linear measurements of embryonic structures].

PubMed

Severtsova, E A; Severtsov, A S

2011-01-01

Investigations of individual variability have allowed us to reveal the crucial (= nodal) stages in embryogenesis of the moor frog (Rana arvalis Nills.). These crucial stages are: the late gastrula stage (stages 18-20), the hatching stages (stages 32-33) and, apparently, early metamorphosis (stage 39). Moreover, we have found that each embryonic structure passes through its specific crucial stages. For example, stage 34 is crucial for the trait "tail width" but is internodal for all other embryonic traits. At this stage, larva passes from an attached to a free-swimming life style. We also found considerable differences between the different frog populations in the the level of developmental variability. These differences were associated with internodal developmental stages.

OMA-gosh, where's that TAF?

PubMed

Blackwell, T Keith; Walker, Amy K

2008-10-03

How transcription is silenced in early embryos has long been a mystery. In this issue, Guven-Ozkan et al. (2008) report that transcriptional repression during worm embryogenesis is mediated through sequestration of the general transcription factor TAF-4 and is regulated by mechanisms that orchestrate the transition between maternal and zygotic gene expression.

Recent advances in the study of somatic mosaicism and diseases other than cancer.

PubMed

Erickson, Robert P

2014-06-01

Somatic mosaicism is well appreciated as a cause of cancer and, possibly, aging. Somatic mosaicism as the cause of other diseases is becoming more appreciated. It is especially important in the causation of deforming diseases (e.g., Proteus syndrome; Sturge-Weber syndrome) which are not inherited because early developmental expression is lethal. It also known to make an important contribution to neurological, dermatological, hematological and other diseases (and probably all diseases but many in which it is harder to detect). There have been exciting recent advances in the detection of somatic mosaicism. In particular, for many diseases of somatic overgrowth in which somatic mosaicism as the sole cause was predicted, the gene somatically mutated has been found. A limited number of pathways seem involved in these disorders, some of which are also implicated in cancer. Copyright © 2014 Elsevier Ltd. All rights reserved.

MICROSPOROGENESIS AND EMBRYOGENESIS IN QUERCUS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stairs, G. R.

1962-01-01

Representative species from two subgenera in the genus Quercus were examined for floral structure and phenology, microsporogenesis, and embryogenesis. The species selected for investigation were: Quercus alba in the Lepidobalanus subgenera, and Quercus coccinea and Quercus ilicifolia from the Erythrobalanus group. Photographs of flowering and photomicrographs of microsporogensis and embryogenesis are used for illustration. The male flowers of the three species are borne on catkins which develop in the scale leaf axils of the current vegetative bud or in separate male buds. Meiosis occurred in the spring at the beginning of bud enlargement; division figures were regular in all themore » material observed. A haploid chromosome number of 12 was confirmed for the three species. Pollen was shed on May 10, 1962, from trees of Quercus coccinea and Quercus ilicifolia; and on May 26, 1962 from Quercus alba. The female flowers are located in the axils of the new leaves. Seed development requires one growing season in Quercus alba, but two growing seasons are required to mature seed of Quercus coccinea and Quercus ilicifolia. The chronology of embryo development was similar for Quercus coccinea and Quercus ilicifolia; embryo development of Quercus alba was about two weeks behind that of the other two species. Definition of ovule dominance within a seed occurred at the time of early embryo development. Failure of this physiological expression of dominance results in multiseeded acorns. No abnormal embryogenesis per se was observed in relation to multiple embryo development. (auth)« less

Differential gene expression during early embryonic development in diapause and non-diapause eggs of multivoltine silkworm Bombyx mori.

PubMed

Ponnuvel, Kangayam M; Murthy, Geetha N; Awasthi, Arvind K; Rao, Guruprasad; Vijayaprakash, Nanjappa B

2010-11-01

Quantification of the differential expression of metabolic enzyme and heat-shock protein genes (Hsp) during early embryogenesis in diapause and non-diapause eggs of the silkworm B. mori was carried out by semi-quantitative RT-PCR. Data analysis revealed that, the phosphofructokinase (PFK) expression started at a higher level in the early stage (6 h after oviposition) in non-diapause eggs, while in diapause induced eggs, it started at a lower level. However, the PFK gene expression in diapause eggs was comparatively higher than in non-diapause eggs. PFK facilitates use of carbohydrate reserves. The lower level of PFK gene expression in the early stage of diapause induced eggs but comparatively higher level of expression than in non-diapause eggs is due to enzyme inactivation via protein phosphorylation during early embryogenesis followed by de-phosphorylation in later stage. The sorbitol dehydrogenase-2 (SDH-2) gene was down regulated in diapause induced eggs up to 24 h and its expression levels in diapause induced eggs coincided with that of PFK gene at 48h in non-diapause eggs. During carbohydrate metabolism, there is an initial temporary accumulation of sorbitol which acts as protectant. The down regulation of SDH-2 gene during the first 24 hours in diapause induced eggs was due to the requirement of sorbitol as protectant. However, since the diapause process culminates by 48 h, the SDH-2 gene expression increased and coincided with that of PFK gene expression. The trehalase (Tre) gene expression was at a lower level in diapause induced eggs compared to non-diapausing eggs. The induction of Tre activity is to regulate uptake and use of sugar by the tissues. The non-diapause eggs revealed maximum expression of GPase gene with major fluctuations as well as an overall higher expression compared to diapause induced eggs. The diapause process requires less energy source which reflects lower activity of the gene. Heat shock protein (Hsp) genes (Hsp20.4, 40, 70, and 90) revealed differential levels of expression in both the eggs at all stages of embryonic development. The present study thus provides an overview of the differential expression levels of metabolic enzyme and Hsp genes in non-diapause and diapause induced eggs of multivoltine silkworm B. mori within 48 h after oviposition, confirming the major role of in early embryogenesis.

Wolbachia as a bacteriocyte-associated nutritional mutualist

PubMed Central

Hosokawa, Takahiro; Koga, Ryuichi; Kikuchi, Yoshitomo; Meng, Xian-Ying; Fukatsu, Takema

2009-01-01

Many insects are dependent on bacterial symbionts that provide essential nutrients (ex. aphid–Buchnera and tsetse–Wiglesworthia associations), wherein the symbionts are harbored in specific cells called bacteriocytes that constitute a symbiotic organ bacteriome. Facultative and parasitic bacterial symbionts like Wolbachia have been regarded as evolutionarily distinct from such obligate nutritional mutualists. However, we discovered that, in the bedbug Cimex lectularius, Wolbachia resides in a bacteriome and appears to be an obligate nutritional mutualist. Two bacterial symbionts, a Wolbachia strain and an unnamed γ-proteobacterium, were identified from different strains of the bedbug. The Wolbachia symbiont was detected from all of the insects examined whereas the γ-proteobacterium was found in a part of them. The Wolbachia symbiont was specifically localized in the bacteriomes and vertically transmitted via the somatic stem cell niche of germalia to oocytes, infecting the incipient symbiotic organ at an early stage of the embryogenesis. Elimination of the Wolbachia symbiont resulted in retarded growth and sterility of the host insect. These deficiencies were rescued by oral supplementation of B vitamins, confirming the essential nutritional role of the symbiont for the host. The estimated genome size of the Wolbachia symbiont was around 1.3 Mb, which was almost equivalent to the genome sizes of parasitic Wolbachia strains of other insects. These results indicate that bacteriocyte-associated nutritional mutualism can evolve from facultative and prevalent microbial associates like Wolbachia, highlighting a previously unknown aspect of the parasitism-mutualism evolutionary continuum. PMID:20080750

Untangling the Contributions of Sex-Specific Gene Regulation and X-Chromosome Dosage to Sex-Biased Gene Expression in Caenorhabditis elegans

PubMed Central

Kramer, Maxwell; Rao, Prashant; Ercan, Sevinc

2016-01-01

Dosage compensation mechanisms equalize the level of X chromosome expression between sexes. Yet the X chromosome is often enriched for genes exhibiting sex-biased, i.e., imbalanced expression. The relationship between X chromosome dosage compensation and sex-biased gene expression remains largely unexplored. Most studies determine sex-biased gene expression without distinguishing between contributions from X chromosome copy number (dose) and the animal’s sex. Here, we uncoupled X chromosome dose from sex-specific gene regulation in Caenorhabditis elegans to determine the effect of each on X expression. In early embryogenesis, when dosage compensation is not yet fully active, X chromosome dose drives the hermaphrodite-biased expression of many X-linked genes, including several genes that were shown to be responsible for hermaphrodite fate. A similar effect is seen in the C. elegans germline, where X chromosome dose contributes to higher hermaphrodite X expression, suggesting that lack of dosage compensation in the germline may have a role in supporting higher expression of X chromosomal genes with female-biased functions in the gonad. In the soma, dosage compensation effectively balances X expression between the sexes. As a result, somatic sex-biased expression is almost entirely due to sex-specific gene regulation. These results suggest that lack of dosage compensation in different tissues and developmental stages allow X chromosome copy number to contribute to sex-biased gene expression and function. PMID:27356611

Spatial and temporal plasticity of chromatin during programmed DNA-reorganization in Stylonychia macronuclear development

PubMed Central

Postberg, Jan; Heyse, Katharina; Cremer, Marion; Cremer, Thomas; Lipps, Hans J

2008-01-01

Background: In this study we exploit the unique genome organization of ciliates to characterize the biological function of histone modification patterns and chromatin plasticity for the processing of specific DNA sequences during a nuclear differentiation process. Ciliates are single-cell eukaryotes containing two morphologically and functionally specialized types of nuclei, the somatic macronucleus and the germline micronucleus. In the course of sexual reproduction a new macronucleus develops from a micronuclear derivative. During this process specific DNA sequences are eliminated from the genome, while sequences that will be transcribed in the mature macronucleus are retained. Results: We show by immunofluorescence microscopy, Western analyses and chromatin immunoprecipitation (ChIP) experiments that each nuclear type establishes its specific histone modification signature. Our analyses reveal that the early macronuclear anlage adopts a permissive chromatin state immediately after the fusion of two heterochromatic germline micronuclei. As macronuclear development progresses, repressive histone modifications that specify sequences to be eliminated are introduced de novo. ChIP analyses demonstrate that permissive histone modifications are associated with sequences that will be retained in the new macronucleus. Furthermore, our data support the hypothesis that a PIWI-family protein is involved in a transnuclear cross-talk and in the RNAi-dependent control of developmental chromatin reorganization. Conclusion: Based on these data we present a comprehensive analysis of the spatial and temporal pattern of histone modifications during this nuclear differentiation process. Results obtained in this study may also be relevant for our understanding of chromatin plasticity during metazoan embryogenesis. PMID:19014664

Redeployment of germ layers related TFs shows regionalized expression during two non-embryonic developments.

PubMed

Ricci, Lorenzo; Cabrera, Fabien; Lotito, Sonia; Tiozzo, Stefano

2016-08-01

In all non-vertebrate metazoan phyla, species that evolved non-embryonic developmental pathways as means of propagation or regeneration can be found. In this context, new bodies arise through asexual reproduction processes (such as budding) or whole body regeneration, that lack the familiar temporal and spatial cues classically associated with embryogenesis, like maternal determinants, or gastrulation. The molecular mechanisms underlying those non-embryonic developments (i.e., regeneration and asexual reproduction), and their relationship to those deployed during embryogenesis are poorly understood. We have addressed this question in the colonial ascidian Botryllus schlosseri, which undergoes an asexual reproductive process via palleal budding (PB), as well as a whole body regeneration by vascular budding (VB). We identified early regenerative structures during VB and then followed the fate of differentiating tissues during both non-embryonic developments (PB and VB) by monitoring the expression of genes known to play key functions in germ layer specification with well conserved expression patterns in solitary ascidian embryogenesis. The expression patterns of FoxA1, GATAa, GATAb, Otx, Bra, Gsc and Tbx2/3 were analysed during both PB and VB. We found that the majority of these transcription factors were expressed during both non-embryonic developmental processes, revealing a regionalization of the palleal and vascular buds. Knockdown of GATAa by siRNA in palleal buds confirmed that preventing the correct development of one of these regions blocks further tissue specification. Our results indicate that during both normal and injury-induced budding, a similar alternative developmental program operates via early commitment of epithelial regions. Copyright © 2016. Published by Elsevier Inc.

Evaluating biomechanical properties of murine embryos using Brillouin microscopy and optical coherence tomography

NASA Astrophysics Data System (ADS)

Raghunathan, Raksha; Zhang, Jitao; Wu, Chen; Rippy, Justin; Singh, Manmohan; Larin, Kirill V.; Scarcelli, Giuliano

2017-08-01

Embryogenesis is regulated by numerous changes in mechanical properties of the cellular microenvironment. Thus, studying embryonic mechanophysiology can provide a more thorough perspective of embryonic development, potentially improving early detection of congenital abnormalities as well as evaluating and developing therapeutic interventions. A number of methods and techniques have been used to study cellular biomechanical properties during embryogenesis. While some of these techniques are invasive or involve the use of external agents, others are compromised in terms of spatial and temporal resolutions. We propose the use of Brillouin microscopy in combination with optical coherence tomography (OCT) to measure stiffness as well as structural changes in a developing embryo. While Brillouin microscopy assesses the changes in stiffness among different organs of the embryo, OCT provides the necessary structural guidance.

Differential expression of two scribble isoforms during Drosophila embryogenesis.

PubMed

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

2001-10-01

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

The impact of transposable elements on mammalian development.

PubMed

Garcia-Perez, Jose L; Widmann, Thomas J; Adams, Ian R

2016-11-15

Despite often being classified as selfish or junk DNA, transposable elements (TEs) are a group of abundant genetic sequences that have a significant impact on mammalian development and genome regulation. In recent years, our understanding of how pre-existing TEs affect genome architecture, gene regulatory networks and protein function during mammalian embryogenesis has dramatically expanded. In addition, the mobilization of active TEs in selected cell types has been shown to generate genetic variation during development and in fully differentiated tissues. Importantly, the ongoing domestication and evolution of TEs appears to provide a rich source of regulatory elements, functional modules and genetic variation that fuels the evolution of mammalian developmental processes. Here, we review the functional impact that TEs exert on mammalian developmental processes and discuss how the somatic activity of TEs can influence gene regulatory networks. © 2016. Published by The Company of Biologists Ltd.

Copy number variation is a fundamental aspect of the placental genome.

PubMed

Hannibal, Roberta L; Chuong, Edward B; Rivera-Mulia, Juan Carlos; Gilbert, David M; Valouev, Anton; Baker, Julie C

2014-05-01

Discovery of lineage-specific somatic copy number variation (CNV) in mammals has led to debate over whether CNVs are mutations that propagate disease or whether they are a normal, and even essential, aspect of cell biology. We show that 1,000 N polyploid trophoblast giant cells (TGCs) of the mouse placenta contain 47 regions, totaling 138 Megabases, where genomic copies are underrepresented (UR). UR domains originate from a subset of late-replicating heterochromatic regions containing gene deserts and genes involved in cell adhesion and neurogenesis. While lineage-specific CNVs have been identified in mammalian cells, classically in the immune system where V(D)J recombination occurs, we demonstrate that CNVs form during gestation in the placenta by an underreplication mechanism, not by recombination nor deletion. Our results reveal that large scale CNVs are a normal feature of the mammalian placental genome, which are regulated systematically during embryogenesis and are propagated by a mechanism of underreplication.

RNA Interference for Functional Genomics and Improvement of Cotton (Gossypium sp.)

PubMed Central

Abdurakhmonov, Ibrokhim Y.; Ayubov, Mirzakamol S.; Ubaydullaeva, Khurshida A.; Buriev, Zabardast T.; Shermatov, Shukhrat E.; Ruziboev, Haydarali S.; Shapulatov, Umid M.; Saha, Sukumar; Ulloa, Mauricio; Yu, John Z.; Percy, Richard G.; Devor, Eric J.; Sharma, Govind C.; Sripathi, Venkateswara R.; Kumpatla, Siva P.; van der Krol, Alexander; Kater, Hake D.; Khamidov, Khakimdjan; Salikhov, Shavkat I.; Jenkins, Johnie N.; Abdukarimov, Abdusattor; Pepper, Alan E.

2016-01-01

RNA interference (RNAi), is a powerful new technology in the discovery of genetic sequence functions, and has become a valuable tool for functional genomics of cotton (Gossypium sp.). The rapid adoption of RNAi has replaced previous antisense technology. RNAi has aided in the discovery of function and biological roles of many key cotton genes involved in fiber development, fertility and somatic embryogenesis, resistance to important biotic and abiotic stresses, and oil and seed quality improvements as well as the key agronomic traits including yield and maturity. Here, we have comparatively reviewed seminal research efforts in previously used antisense approaches and currently applied breakthrough RNAi studies in cotton, analyzing developed RNAi methodologies, achievements, limitations, and future needs in functional characterizations of cotton genes. We also highlighted needed efforts in the development of RNAi-based cotton cultivars, and their safety and risk assessment, small and large-scale field trials, and commercialization. PMID:26941765

Concepts, strategies and potentials using hypo-g and other features of the space environment for commercialization using higher plants

NASA Technical Reports Server (NTRS)

Krikorian, A. D.

1985-01-01

Opportunities for releasing, capturing, constructing and/or fixing the differential expressions or response potentials of the higher plant genome in the hypo-g environment for commercialization are explored. General strategies include improved plant-growing, crop and forestry production systems which conserve soil, water, labor and energy resources, and nutritional partitioning and mobilization of nutrients and synthates. Tissue and cell culture techniques of commercial potential include the growing and manipulation of cultured plant cells in vitro in a bioreactor to produce biologicals and secondary plants of economic value. The facilitation of plant breeding, the cloning of specific pathogen-free materials, the elimination of growing point or apex viruses, and the increase of plant yield are other O-g applications. The space environment may be advantageous in somatic embryogenesis, the culture of alkaloids, and the development of completely new crop plant germ plasm.

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.

Loss of the tumor suppressor BAP1 causes myeloid transformation

PubMed Central

Dey, Anwesha; Seshasayee, Dhaya; Noubade, Rajkumar; French, Dorothy M.; Liu, Jinfeng; Chaurushiya, Mira S.; Kirkpatrick, Donald S.; Pham, Victoria C.; Lill, Jennie R.; Bakalarski, Corey E.; Wu, Jiansheng; Phu, Lilian; Katavolos, Paula; Saunders, Lindsay M.; Abdel-Wahab, Omar; Modrusan, Zora; Seshagiri, Somasekar; Dong, Ken; Lin, Zhonghua; Balazs, Mercedesz; Suriben, Rowena; Newton, Kim; Hymowitz, Sarah; Garcia-Manero, Guillermo; Martin, Flavius; Levine, Ross L.; Dixit, Vishva M.

2016-01-01

Deubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with increased risk of mesothelioma and uveal melanoma. Somatic BAP1 mutations occur in various malignancies. We show that mouse Bap1 gene deletion is lethal during embryogenesis, but systemic or hematopoietic-restricted deletion in adults recapitulates features of human myelodysplastic syndrome (MDS). Knock-in mice expressing BAP1 with a 3xFlag tag revealed that BAP1 interacts with HCF-1, OGT, and the polycomb group proteins ASXL1 and ASXL2 in vivo. OGT and HCF-1 levels were decreased by Bap1 deletion, indicating a critical role for BAP1 in stabilizing these epigenetic regulators. Human ASXL1 is mutated frequently in chronic myelomonocytic leukemia (CMML) so an ASXL/BAP1 complex may suppress CMML. A novel BAP1 catalytic mutation found in a MDS patient implies that BAP1 loss of function has similar consequences in mouse and man. PMID:22878500

RNA Interference for Functional Genomics and Improvement of Cotton (Gossypium sp.).

PubMed

Abdurakhmonov, Ibrokhim Y; Ayubov, Mirzakamol S; Ubaydullaeva, Khurshida A; Buriev, Zabardast T; Shermatov, Shukhrat E; Ruziboev, Haydarali S; Shapulatov, Umid M; Saha, Sukumar; Ulloa, Mauricio; Yu, John Z; Percy, Richard G; Devor, Eric J; Sharma, Govind C; Sripathi, Venkateswara R; Kumpatla, Siva P; van der Krol, Alexander; Kater, Hake D; Khamidov, Khakimdjan; Salikhov, Shavkat I; Jenkins, Johnie N; Abdukarimov, Abdusattor; Pepper, Alan E

2016-01-01

RNA interference (RNAi), is a powerful new technology in the discovery of genetic sequence functions, and has become a valuable tool for functional genomics of cotton (Gossypium sp.). The rapid adoption of RNAi has replaced previous antisense technology. RNAi has aided in the discovery of function and biological roles of many key cotton genes involved in fiber development, fertility and somatic embryogenesis, resistance to important biotic and abiotic stresses, and oil and seed quality improvements as well as the key agronomic traits including yield and maturity. Here, we have comparatively reviewed seminal research efforts in previously used antisense approaches and currently applied breakthrough RNAi studies in cotton, analyzing developed RNAi methodologies, achievements, limitations, and future needs in functional characterizations of cotton genes. We also highlighted needed efforts in the development of RNAi-based cotton cultivars, and their safety and risk assessment, small and large-scale field trials, and commercialization.

[Current progress and future direction in the biology of ovarian germ stem cells in mammals].

PubMed

Li, Chao-Hui; Guo, Kun; Zheng, Ping

2012-12-01

Whether or not oogenesis continues after birth in mammalian ovaries remains controversial. Since the 1950's, it has been generally accepted that oogenesis takes place during embryogenesis in mammals and ceases at birth. At birth, germ cells in mammalian ovaries have progressed to the diplotene stage of meiotic prophase and have formed primordial follicles with surrounding somatic cells. These primordial follicles represent follicle reserves of the reproductive life. However, this view has been recently challenged by a growing body of evidence showing the isolation and propagation of germ stem cells from mouse and human ovaries. These ovarian germ stem cells are capable of regenerating functional oocytes when transplanted back into recipient ovaries. Despite the discovery of the potential germ stem cells in mammalian ovaries, it remains uncertain whether these cells exist and function in ovaries under physiological conditions. Herein we review the current progress and future direction in this infant area.

TDZ pulsing evaluation on the in vitro morphogenesis of peach palm.

PubMed

Graner, Erika Mendes; Oberschelp, Gustavo Pedro Javier; Brondani, Gilvano Ebling; Batagin-Piotto, Katherine Derlene; de Almeida, Cristina Vieira; de Almeida, Marcílio

2013-04-01

Peach palm (Bactris gasipaes Kunth.) cropping is an excellent alternative to native species exploitation; nevertheless, the problems with seed germination and conventional propagation justify the use of in vitro culturing. Aiming to asses TDZ pulsing effect on B. gasipaes morphogenesis, explants obtained from unarmed microplants were maintained in two treatments, half of them in MS free medium (without growth regulator) and the other half in MS with TDZ (0.36 μM). Both groups were transferred to growth regulator-free MS medium following 14 days of culture. After 84 days of culture, TDZ pulsing increased the growth and development of the shoots, restricted the growth and development of the roots, with no influence on adventitious bud induction or somatic embryogenesis. Furthermore, development of prickles, thickening of roots and chlorotic leaves were noted under TDZ pulsing. Leaf sheath histological analysis showed an epidermal origin and no vascularization of these prickles.

Pepper, sweet (Capsicum annuum).

PubMed

Heidmann, Iris; Boutilier, Kim

2015-01-01

Capsicum (pepper) species are economically important crops that are recalcitrant to genetic transformation by Agrobacterium (Agrobacterium tumefaciens). A number of protocols for pepper transformation have been described but are not routinely applicable. The main bottleneck in pepper transformation is the low frequency of cells that are both susceptible for Agrobacterium infection and have the ability to regenerate. Here, we describe a protocol for the efficient regeneration of transgenic sweet pepper (C. annuum) through inducible activation of the BABY BOOM (BBM) AP2/ERF transcription factor. Using this approach, we can routinely achieve a transformation efficiency of at least 0.6 %. The main improvements in this protocol are the reproducibility in transforming different genotypes and the ability to produce fertile shoots. An added advantage of this protocol is that BBM activity can be induced subsequently in stable transgenic lines, providing a novel regeneration system for clonal propagation through somatic embryogenesis.

EVALUATION OF FERTILIZATION FOLLOWING OVULATORY DELAY WITH THIRAM IN THE LONG-EVANS HOODED RAT

EPA Science Inventory

Evaluation of fertilization following ovulatory delay with thiram in the Long-Evans Hooded Rat

1TE Stoker, 1* S Jeffay, and 1 SD Perreault.

1Gamete and Early Embryogenesis Biology Branch and 2 Endocrinology Branch, Reproductive Toxicology Division, NHEERL, US EPA, R...

Four queries concerning the metaphysics of early human embryogenesis.

PubMed

Howsepian, A A

2008-04-01

In this essay, I attempt to provide answers to the following four queries concerning the metaphysics of early human embryogenesis. (1) Following its first cellular fission, is it coherent to claim that one and only one of two "blastomeric" twins of a human zygote is identical with that zygote? (2) Following the fusion of two human pre-embryos, is it coherent to claim that one and only one pre-fusion pre-embryo is identical with that postfusion pre-embryo? (3) Does a live human being come into existence only when its brain comes into existence? (4) At implantation, does a pre-embryo become a mere part of its mother? I argue that either if things have quidditative properties or if criterialism is false, then queries (1) and (2) can be answered in the affirmative; that in light of recent developments in theories of human death and in light of a more "functional" theory of brains, query (3) can be answered in the negative; and that plausible mereological principles require a negative answer to query (4).

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.

Regeneration of viable oil palm plants from protoplasts by optimizing media components, growth regulators and cultivation procedures.

PubMed

Masani, Mat Yunus Abdul; Noll, Gundula; Parveez, Ghulam Kadir Ahmad; Sambanthamurthi, Ravigadevi; Prüfer, Dirk

2013-09-01

Oil palm protoplasts are suitable as a starting material for the production of oil palm plants with new traits using approaches such as somatic hybridization, but attempts to regenerate viable plants from protoplasts have failed thus far. Here we demonstrate, for the first time, the regeneration of viable plants from protoplasts isolated from cell suspension cultures. We achieved a protoplast yield of 1.14×10(6) per gram fresh weight with a viability of 82% by incubating the callus in a digestion solution comprising 2% cellulase, 1% pectinase, 0.5% cellulase onuzuka R10, 0.1% pectolyase Y23, 3% KCl, 0.5% CaCl2 and 3.6% mannitol. The regeneration of protoplasts into viable plants required media optimization, the inclusion of plant growth regulators and the correct culture technique. Microcalli derived from protoplasts were obtained by establishing agarose bead cultures using Y3A medium supplemented with 10μM naphthalene acetic acid, 2μM 2,4-dichlorophenoxyacetic acid, 2μM indole-3-butyric acid, 2μM gibberellic acid and 2μM 2-γ-dimethylallylaminopurine. Small plantlets were regenerated from microcalli by somatic embryogenesis after successive subculturing steps in medium with limiting amounts of growth regulators supplemented with 200mg/l ascorbic acid. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

A LINE-1 Component to Human Aging: Do LINE elements exact a longevity cost for evolutionary advantage?

PubMed Central

Laurent, Georges St.; Hammell, Neil; McCaffrey, Timothy A.

2010-01-01

Advancing age remains the largest risk factor for devastating diseases, such as heart disease, stroke, and cancer. The mechanisms by which advancing age predisposes to disease are now beginning to unfold, due in part, to genetic and environmental manipulations of longevity in lower organisms. Converging lines of evidence suggest that DNA damage may be a final common pathway linking several proposed mechanisms of aging. The present review forwards a theory for an additional aging pathway that involves modes of inherent genetic instability. Long interspersed nuclear elements (LINEs) are endogenous non-LTR retrotransposons that compose about 20% of the human genome. The LINE-1 (L1) gene products, ORF1p and ORF2p, possess mRNA binding, endonuclease, and reverse transcriptase activity that enable retrotransposition. While principally active only during embryogenesis, L1 transcripts are detected in adult somatic cells under certain conditions. The present hypothesis proposes that L1s act as an ‘endogenous clock’, slowly eroding genomic integrity by competing with the organism’s double-strand break repair mechanism. Thus, while L1s are an accepted mechanism of genetic variation fueling evolution, it is proposed that longevity is negatively impacted by somatic L1 activity. The theory predicts testable hypotheses about the relationship between L1 activity, DNA repair, healthy aging, and longevity. PMID:20346965

Characterizing early embryonic development of Brown Tsaiya Ducks (Anas platyrhynchos) in comparison with Taiwan Country Chicken (Gallus gallus domestics)

PubMed Central

Lumsangkul, Chompunut; Fan, Yang-Kwang; Chang, Shen-Chang; Ju, Jyh-Cherng

2018-01-01

Avian embryos are among the most convenient and the primary representatives for the study of classical embryology. It is well-known that the hatching time of duck embryos is approximately one week longer than that of chicken embryos. However, the key features associated with the slower embryonic development in ducks have not been adequately described. This study aimed to characterize the pattern and the speed of early embryogenesis in Brown Tsaiya Ducks (BTD) compared with those in Taiwan Country Chicken (TCC) by using growth parameters including embryonic crown-tail length (ECTL), primitive streak formation, somitogenesis, and other development-related parameters, during the first 72 h of incubation. Three hundred and sixty eggs from BTD and TCC, respectively, were incubated at 37.2°C, and were then dissected hourly to evaluate their developmental stages. We found that morphological changes of TCC embryos shared a major similarity with that of the Hamburger and Hamilton staging system during early chick embryogenesis. The initial primitive streak in TCC emerged between 6 and 7 h post-incubation, but its emergence was delayed until 10 to 13 h post-incubation in BTD. Similarly, the limb primordia (wing and limb buds) were observed at 51 h post-incubation in TCC embryos compared to 64 h post-incubation in BTD embryos. The allantois first appeared around 65 to 68 h in TCC embryos, but it was not observed in BTD embryos. At the 72 h post-incubation, 40 somites were clearly formed in TCC embryos while only 32 somites in BTD embryos. Overall, the BTD embryos developed approximately 16 h slower than the chicken embryo during the first 72 h of development. To our best knowledge, this is the first study to describe two distinct developmental time courses between TCC and BTD, which would facilitate future embryogenesis-related studies of the two important avian species in Taiwan. PMID:29742160

The conceptual framework of evolutionary morphology in the studies of Ernst Haeckel and Fritz Müller.

PubMed

Breidbach, Olaf

2006-03-01

In his Gastraea studies Ernst Haeckel characterized the initial stages of the animal embryo, describing complete and incomplete cleavages in various groups, until the gastrula stage. Thereby, he was able to point out various degrees of developmental diversification in these initial stages of development. As the functional meaning of such cleavages was not clear however, it was difficult to argue about putative functional adaptations. Information about the consequences for tissue formation initiated in this primary phase of development was simply lacking. Haeckel could only provide a vague picture of a highly diversified but systematically inconsistent distribution of various types of early embryogenesis. Thereby he discusses phylogenetically preserved (palingenetic) stages of development and adaptations to certain specific situations of the embryo (cenogenesis). To decide whether such types, in the initial stages of embryogenesis, are ceno- or phaenogenetic is quite difficult. Reference to the highly diversified distribution of certain types within specific groups is an indication that there is no strict adaptive pressure on these early parts of embryonic development. This makes it possible to formulate - as Haeckel did it - the idea, that in these initial phases palingenetic attributes are dominant. Thus, he tried to use these early phases of development for the classification of larger systematic units. The result is a concept of an evolutionary morphology, that was, however, never elaborated in detail by Haeckel. Therefore, it remained without effect for evolutionary biology. On the contrary, following the Darwinian approach towards a comparative analysis of embryogenesis, Fritz Müller presented a series of examples for a comparative developmental biology that allowed one to interpret certain morphological characteristics as the outcome of common evolutionary histories within different species. For various crustacean species, he was able to demonstrate that certain attributes are not to be characterized as functionally relevant adaptations, but are evolutionarily inherited.

Analyses of Sox-B and Sox-E Family Genes in the Cephalopod Sepia officinalis: Revealing the Conserved and the Unusual

PubMed Central

Focareta, Laura; Cole, Alison G.

2016-01-01

Cephalopods provide an unprecedented opportunity for comparative studies of the developmental genetics of organ systems that are convergent with analogous vertebrate structures. The Sox-family of transcription factors is an important class of DNA-binding proteins that are known to be involved in many aspects of differentiation, but have been largely unstudied in lophotrochozoan systems. Using a degenerate primer strategy we have isolated coding sequence for three members of the Sox family of transcription factors from a cephalopod mollusk, the European cuttlefish Sepia officinalis: Sof-SoxE, Sof-SoxB1, and Sof-SoxB2. Analyses of their expression patterns during organogenesis reveals distinct spatial and temporal expression domains. Sof-SoxB1 shows early ectodermal expression throughout the developing epithelium, which is gradually restricted to presumptive sensory epithelia. Expression within the nervous system appears by mid-embryogenesis. Sof-SoxB2 expression is similar to Sof-SoxB1 within the developing epithelia in early embryogenesis, however appears in largely non-overlapping expression domains within the central nervous system and is not expressed in the maturing sensory epithelium. In contrast, Sof-SoxE is expressed throughout the presumptive mesodermal territories at the onset of organogenesis. As development proceeds, Sof-SoxE expression is elevated throughout the developing peripheral circulatory system. This expression disappears as the circulatory system matures, but expression is maintained within undifferentiated connective tissues throughout the animal, and appears within the nervous system near the end of embryogenesis. SoxB proteins are widely known for their role in neural specification in numerous phylogenetic lineages. Our data suggests that Sof-SoxB genes play similar roles in cephalopods. In contrast, Sof-SoxE appears to be involved in the early stages of vasculogenesis of the cephalopod closed circulatory system, a novel role for a member of this gene family. PMID:27331398

Identification of positional candidates for bovine placental genes responsible for early embryonic death during cloning-attempted pregnancy.

PubMed

Yamada, Takahisa; Muramatsu, Youji; Taniguchi, Yukio; Sasaki, Yoshiyuki

Our previous study detected 291 and 77 genes showing early embryonic death-associated elevation and reduction of expression, respectively, in the fetal placenta of the cow carrying somatic nuclear transfer-derived cloned embryo. In this study, we mapped the 10 genes showing the elevation and the 10 genes doing the reduction most significantly, using somatic cell hybrid and bovine draft genome sequence. We then compared the mapped positions for these genes with the genomic locations of bovine quantitative trait loci for still-birth and/or abortion. Among the mapped genes, peptidylglycine alpha-amidating monooxygenase (PAM), spectrin, beta, nonerythrocytic 1 (SPTBNI), and an unknown novel gene containing AU277832 expressed sequence tag were intriguing, in that the mapped positions were consistent with the genomic locations of bovine still-birth and/or abortion quantitative trait loci, and thus identified as positional candidates for bovine placental genes responsible for the early embryonic death during the pregnancy attempted by somatic nuclear transfer-derived cloning.

Somatic symptoms, peer and school stress, and family and community violence exposure among urban elementary school children

PubMed Central

Hart, Shayla L.; Hodgkinson, Stacy C.; Hyman, Corine; Cooley-Strickland, Michele

2013-01-01

Somatic symptoms are a common physical response to stress and illness in childhood. This study assessed 409, primarily African American (85.6 %), urban elementary school children to examine the association between: (1) somatic symptoms and potential external stressors (school and peer stress, family conflict, and community violence) and (2) parent and child agreement on children’s self-report of somatic symptoms. The odds of self-report of somatic complaints were significantly associated with family conflict, school and peer stress, and community violence exposure (OR = 1.26, 95 % CI: 1.05–1.50; OR = 1.18, 95 % CI 1.08–1.28; and OR = 1.02, 95 % CI: 1.00–1.05, respectively). Identifying the associations between social, family, and community based stress and somatic symptoms may improve the quality of life for children living in urban environments through early identification and treatment. PMID:22772584

Somatic symptoms, peer and school stress, and family and community violence exposure among urban elementary school children.

PubMed

Hart, Shayla L; Hodgkinson, Stacy C; Belcher, Harolyn M E; Hyman, Corine; Cooley-Strickland, Michele

2013-10-01

Somatic symptoms are a common physical response to stress and illness in childhood. This study assessed 409, primarily African American (85.6 %), urban elementary school children to examine the association between: (1) somatic symptoms and potential external stressors (school and peer stress, family conflict, and community violence) and (2) parent and child agreement on children's self-report of somatic symptoms. The odds of self-report of somatic complaints were significantly associated with family conflict, school and peer stress, and community violence exposure (OR = 1.26, 95 % CI: 1.05-1.50; OR = 1.18, 95 % CI 1.08-1.28; and OR = 1.02, 95 % CI: 1.00-1.05, respectively). Identifying the associations between social, family, and community based stress and somatic symptoms may improve the quality of life for children living in urban environments through early identification and treatment.

[Formation of antioxidant defence system of geese in embryogenesis and early postnatal ontogenesis].

PubMed

Danchenko, O O; Kalytka, V V

2002-01-01

The features of antioxidant protection of tissues of a liver and blood of the gooses in embriogenesis and early postnatal ontogenesis are found out. Maximal contents TBA active products both in a liver, and in a blood are observed in 28 diurnal embriones. Is shown, that in a liver the activity of basic antioxidant enzymes (superoxide dismutases, catalase and glutathione peroxidase) in a liver is developed already at early stages embriogenesis and is considerably enlarged in the end embriogenesis. The becoming of enzymatic system of a blood descends much more slower.

Arabidopsis thaliana GEX1 has dual functions in gametophyte development and early embryogenesis

USDA-ARS?s Scientific Manuscript database

GEX1 is a plasma membrane protein conserved among plant species, and was previously shown to be expressed in sperm cells and some sporophytic tissues. Here we show that GEX1 is also expressed in the embryo sac before cellularization, in the egg cell after cellularization, in the zygote/embryo immedi...

Non-equivalent contributions of maternal and paternal genomes to early plant embryogenesis.

PubMed

Del Toro-De León, Gerardo; García-Aguilar, Marcelina; Gillmor, C Stewart

2014-10-30

Zygotic genome activation in metazoans typically occurs several hours to a day after fertilization, and thus maternal RNAs and proteins drive early animal embryo development. In plants, despite several molecular studies of post-fertilization transcriptional activation, the timing of zygotic genome activation remains a matter of debate. For example, two recent reports that used different hybrid ecotype combinations for RNA sequence profiling of early Arabidopsis embryo transcriptomes came to divergent conclusions. One identified paternal contributions that varied by gene, but with overall maternal dominance, while the other found that the maternal and paternal genomes are transcriptionally equivalent. Here we assess paternal gene activation functionally in an isogenic background, by performing a large-scale genetic analysis of 49 EMBRYO DEFECTIVE genes and testing the ability of wild-type paternal alleles to complement phenotypes conditioned by mutant maternal alleles. Our results demonstrate that wild-type paternal alleles for nine of these genes are completely functional 2 days after pollination, with the remaining 40 genes showing partial activity beginning at 2, 3 or 5 days after pollination. Using our functional assay, we also demonstrate that different hybrid combinations exhibit significant variation in paternal allele activation, reconciling the apparently contradictory results of previous transcriptional studies. The variation in timing of gene function that we observe confirms that paternal genome activation does not occur in one early discrete step, provides large-scale functional evidence that maternal and paternal genomes make non-equivalent contributions to early plant embryogenesis, and uncovers an unexpectedly profound effect of hybrid genetic backgrounds on paternal gene activity.

Expression of phosphatidylcholine biosynthetic enzymes during early embryogenesis in the amphibian Bufo arenarum.

PubMed

Fernández-Bussy, Rodrigo; Mouguelar, Valeria; Banchio, Claudia; Coux, Gabriela

2015-04-01

In the principal route of phosphatidylcholine (PC) synthesis the regulatory steps are catalysed by CTP:phosphocholine cytidylyltransferase (CCT) and choline kinase (CK). Knock-out mice in Pcyt1a (CCT gene) and Chka1 (CK gene) resulted in preimplantation embryonic lethality, demonstrating the essential role of this pathway. However, there is still a lack of detailed CCT and CK expression analysis during development. The aim of the current work was to study the expression during early development of both enzymes in the external-fertilization vertebrate Bufo arenarum. Reverse transcription polymerase chain reaction (RT-PCR) and western blot confirmed their presence in unfertilized eggs. Analysis performed in total extracts from staged embryos showed constant protein levels of both enzymes until the 32-cell stage: then they decreased, reaching a minimum in the gastrula before starting to recover. CTP:phosphocholine cytidylyltransferase is an amphitropic enzyme that inter-converts between cytosolic inactive and membrane-bound active forms. Immunoblot analysis demonstrated that the cytosolic:total CCT protein ratio does not change throughout embryogenesis, suggesting a progressive decline of CCT activity in early development. However, PC (and phosphatidylethanolamine) content per egg/embryo remained constant throughout the stages analysed. In conclusion, the current data for B. arenarum suggest that net synthesis of PC mediated by CCT and CK is not required in early development and that supplies for membrane biosynthesis are fulfilled by lipids already present in the egg/embryo reservoirs.

Histone variant H3.3-mediated chromatin remodeling is essential for paternal genome activation in mouse preimplantation embryos.

PubMed

Kong, Qingran; Banaszynski, Laura A; Geng, Fuqiang; Zhang, Xiaolei; Zhang, Jiaming; Zhang, Heng; O'Neill, Claire L; Yan, Peidong; Liu, Zhonghua; Shido, Koji; Palermo, Gianpiero D; Allis, C David; Rafii, Shahin; Rosenwaks, Zev; Wen, Duancheng

2018-03-09

Derepression of chromatin-mediated transcriptional repression of paternal and maternal genomes is considered the first major step that initiates zygotic gene expression after fertilization. The histone variant H3.3 is present in both male and female gametes and is thought to be important for remodeling the paternal and maternal genomes for activation during both fertilization and embryogenesis. However, the underlying mechanisms remain poorly understood. Using our H3.3B-HA-tagged mouse model, engineered to report H3.3 expression in live animals and to distinguish different sources of H3.3 protein in embryos, we show here that sperm-derived H3.3 (sH3.3) protein is removed from the sperm genome shortly after fertilization and extruded from the zygotes via the second polar bodies (PBII) during embryogenesis. We also found that the maternal H3.3 (mH3.3) protein is incorporated into the paternal genome as early as 2 h postfertilization and is detectable in the paternal genome until the morula stage. Knockdown of maternal H3.3 resulted in compromised embryonic development both of fertilized embryos and of androgenetic haploid embryos. Furthermore, we report that mH3.3 depletion in oocytes impairs both activation of the Oct4 pluripotency marker gene and global de novo transcription from the paternal genome important for early embryonic development. Our results suggest that H3.3-mediated paternal chromatin remodeling is essential for the development of preimplantation embryos and the activation of the paternal genome during embryogenesis. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

From fibroblasts and stem cells: implications for cell therapies and somatic cloning.

PubMed

Kues, Wilfried A; Carnwath, Joseph W; Niemann, Heiner

2005-01-01

Pluripotent embryonic stem cells (ESCs) from the inner cell mass of early murine and human embryos exhibit extensive self-renewal in culture and maintain their ability to differentiate into all cell lineages. These features make ESCs a suitable candidate for cell-replacement therapy. However, the use of early embryos has provoked considerable public debate based on ethical considerations. From this standpoint, stem cells derived from adult tissues are a more easily accepted alternative. Recent results suggest that adult stem cells have a broader range of potency than imagined initially. Although some claims have been called into question by the discovery that fusion between the stem cells and differentiated cells can occur spontaneously, in other cases somatic stem cells have been induced to commit to various lineages by the extra- or intracellular environment. Recent data from our laboratory suggest that changes in culture conditions can expand a subpopulation of cells with a pluripotent phenotype from primary fibroblast cultures. The present paper critically reviews recent data on the potency of somatic stem cells, methods to modify the potency of somatic cells and implications for cell-based therapies.

Expression profiles of the Gα subunits during Xenopus tropicalis embryonic development.

PubMed

Fuentealba, Jaime; Toro-Tapia, Gabriela; Rodriguez, Marion; Arriagada, Cecilia; Maureira, Alejandro; Beyer, Andrea; Villaseca, Soraya; Leal, Juan I; Hinrichs, Maria V; Olate, Juan; Caprile, Teresa; Torrejón, Marcela

2016-09-01

Heterotrimeric G protein signaling plays major roles during different cellular events. However, there is a limited understanding of the molecular mechanisms underlying G protein control during embryogenesis. G proteins are highly conserved and can be grouped into four subfamilies according to sequence homology and function. To further studies on G protein function during embryogenesis, the present analysis identified four Gα subunits representative of the different subfamilies and determined their spatiotemporal expression patterns during Xenopus tropicalis embryogenesis. Each of the Gα subunit transcripts was maternally and zygotically expressed, and, as development progressed, dynamic expression patterns were observed. In the early developmental stages, the Gα subunits were expressed in the animal hemisphere and dorsal marginal zone. While expression was observed at the somite boundaries, in vascular structures, in the eye, and in the otic vesicle during the later stages, expression was mainly found in neural tissues, such as the neural tube and, especially, in the cephalic vesicles, neural crest region, and neural crest-derived structures. Together, these results support the pleiotropism and complexity of G protein subfamily functions in different cellular events. The present study constitutes the most comprehensive description to date of the spatiotemporal expression patterns of Gα subunits during vertebrate development. Copyright © 2016 Elsevier B.V. All rights reserved.

L1CAM in the Early Enteric and Urogenital System

PubMed Central

Pechriggl, Elisabeth Judith; Concin, Nicole; Blumer, Michael J.; Bitsche, Mario; Zwierzina, Marit; Dudas, Jozsef; Koziel, Katarzyna; Altevogt, Peter; Zeimet, Alain-Gustave; Fritsch, Helga

2016-01-01

L1 cell adhesion molecule (L1CAM) is a transmembrane molecule belonging to the L1 protein family. It has shown to be a key player in axonal guidance in the course of neuronal development. Furthermore, L1CAM is also crucial for the establishment of the enteric and urogenital organs and is aberrantly expressed in cancer originating in these organs. Carcinogenesis and embryogenesis follow a lot of similar molecular pathways, but unfortunately, comprehensive data on L1CAM expression and localization in human developing organs are lacking so far. In the present study we, therefore, examined the spatiotemporal distribution of L1CAM in the early human fetal period (weeks 8–12 of gestation) by means of immunohistochemistry and in situ hybridization (ISH). In the epithelia of the gastrointestinal organs, L1CAM localization cannot be observed in the examined stages most likely due to their advanced polarization and differentiation. Despite these results, our ISH data indicate weak L1CAM expression, but only in few epithelial cells. The genital tracts, however, are distinctly L1CAM positive throughout the entire fetal period. We, therefore, conclude that in embryogenesis L1CAM is crucial for further differentiation of epithelia. PMID:28026654

G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis

PubMed Central

Tachibana, Makoto; Sugimoto, Kenji; Nozaki, Masami; Ueda, Jun; Ohta, Tsutomu; Ohki, Misao; Fukuda, Mikiko; Takeda, Naoki; Niida, Hiroyuki; Kato, Hiroyuki; Shinkai, Yoichi

2002-01-01

Covalent modification of histone tails is crucial for transcriptional regulation, mitotic chromosomal condensation, and heterochromatin formation. Histone H3 lysine 9 (H3-K9) methylation catalyzed by the Suv39h family proteins is essential for establishing the architecture of pericentric heterochromatin. We recently identified a mammalian histone methyltransferase (HMTase), G9a, which has strong HMTase activity towards H3-K9 in vitro. To investigate the in vivo functions of G9a, we generated G9a-deficient mice and embryonic stem (ES) cells. We found that H3-K9 methylation was drastically decreased in G9a-deficient embryos, which displayed severe growth retardation and early lethality. G9a-deficient ES cells also exhibited reduced H3-K9 methylation compared to wild-type cells, indicating that G9a is a dominant H3-K9 HMTase in vivo. Importantly, the loss of G9a abolished methylated H3-K9 mostly in euchromatic regions. Finally, G9a exerted a transcriptionally suppressive function that depended on its HMTase activity. Our results indicate that euchromatic H3-K9 methylation regulated by G9a is essential for early embryogenesis and is involved in the transcriptional repression of developmental genes. PMID:12130538

Identification of Elf-1 and B61 as high affinity ligands for the receptor tyrosine kinase MDK1.

PubMed

Ciossek, T; Ullrich, A

1997-01-09

Mouse Developmental Kinase 1 (MDK1) is a receptor tyrosine kinase of the eck/eph subfamily expressed in a variety of tissues during early mouse embryogenesis. To obtain further insight into the function of MDK1, we determined identity and localisation of its physiological ligand(s). Staining whole embryos with fusion proteins between the extracellular domain of MDK1 and human secreted alkaline phosphatase revealed areas of high receptor binding in the caudal mesencephalon, the frontal neocortex and the limb buds. This staining was sensitive to treatment with phosphatidylinositol-specific phospholipase C. Using Scatchard analysis, high affinity binding of Elf-1 (1.7 x 10(-10) M) and B61 (2.2 x 10(-10) M) towards MDK1 could be demonstrated. However, the transmembrane ligand Lerk2 displayed no measurable affinity for MDK1. Elf-1 and B61 bind to the three full-length MDK1 isoforms with similar dissociation constants. Slightly lower affinities were observed for the two truncated receptors MDK1-Tl and MDK1-T2. The activation of MDK1 with Elf-1 or B61 leads to the rapid autophosphorylation of MDK1 as well as tyrosine phosphorylation of an unknown 62 kDa phosphoprotein in Rat1 cells. These findings implicate MDK1 in patterning processes during early mouse embryogenesis and suggest MDK1 involvement in early organogenesis and midbrain development.

Darker eggs of mosquitoes resist more to dry conditions: Melanin enhances serosal cuticle contribution in egg resistance to desiccation in Aedes, Anopheles and Culex vectors.

PubMed

Farnesi, Luana C; Vargas, Helena C M; Valle, Denise; Rezende, Gustavo L

2017-10-01

Mosquito vectors lay their white eggs in the aquatic milieu. During early embryogenesis water passes freely through the transparent eggshell, which at this moment is composed of exochorion and endochorion. Within two hours the endochorion darkens via melanization but even so eggs shrink and perish if removed from moisture. However, during mid-embryogenesis, cells of the extraembryonic serosa secrete the serosal cuticle, localized right below the endochorion, becoming the third and innermost eggshell layer. Serosal cuticle formation greatly reduces water flow and allows egg survival outside the water. The degree of egg resistance to desiccation (ERD) at late embryogenesis varies among different species: Aedes aegypti, Anopheles aquasalis and Culex quinquefasciatus eggs can survive in a dry environment for ≥ 72, 24 and 5 hours, respectively. In some adult insects, darker-body individuals show greater resistance to desiccation than lighter ones. We asked if egg melanization enhances mosquito serosal cuticle-dependent ERD. Species with higher ERD at late embryogenesis exhibit more melanized eggshells. The melanization-ERD hypothesis was confirmed employing two Anopheles quadrimaculatus strains, the wild type and the mutant GORO, with a dark-brown and a golden eggshell, respectively. In all cases, serosal cuticle formation is fundamental for the establishment of an efficient ERD but egg viability outside the water is much higher in mosquitoes with darker eggshells than in those with lighter ones. The finding that pigmentation influences egg water balance is relevant to understand the evolutionary history of insect egg coloration. Since eggshell and adult cuticle pigmentation ensure insect survivorship in some cases, they should be considered regarding species fitness and novel approaches for vector or pest insects control.

Darker eggs of mosquitoes resist more to dry conditions: Melanin enhances serosal cuticle contribution in egg resistance to desiccation in Aedes, Anopheles and Culex vectors

PubMed Central

Farnesi, Luana C.; Vargas, Helena C. M.; Valle, Denise

2017-01-01

Mosquito vectors lay their white eggs in the aquatic milieu. During early embryogenesis water passes freely through the transparent eggshell, which at this moment is composed of exochorion and endochorion. Within two hours the endochorion darkens via melanization but even so eggs shrink and perish if removed from moisture. However, during mid-embryogenesis, cells of the extraembryonic serosa secrete the serosal cuticle, localized right below the endochorion, becoming the third and innermost eggshell layer. Serosal cuticle formation greatly reduces water flow and allows egg survival outside the water. The degree of egg resistance to desiccation (ERD) at late embryogenesis varies among different species: Aedes aegypti, Anopheles aquasalis and Culex quinquefasciatus eggs can survive in a dry environment for ≥ 72, 24 and 5 hours, respectively. In some adult insects, darker-body individuals show greater resistance to desiccation than lighter ones. We asked if egg melanization enhances mosquito serosal cuticle-dependent ERD. Species with higher ERD at late embryogenesis exhibit more melanized eggshells. The melanization-ERD hypothesis was confirmed employing two Anopheles quadrimaculatus strains, the wild type and the mutant GORO, with a dark-brown and a golden eggshell, respectively. In all cases, serosal cuticle formation is fundamental for the establishment of an efficient ERD but egg viability outside the water is much higher in mosquitoes with darker eggshells than in those with lighter ones. The finding that pigmentation influences egg water balance is relevant to understand the evolutionary history of insect egg coloration. Since eggshell and adult cuticle pigmentation ensure insect survivorship in some cases, they should be considered regarding species fitness and novel approaches for vector or pest insects control. PMID:29084225

The influence of gravity on the process of development of animal systems

NASA Technical Reports Server (NTRS)

Malacinski, G. M.; Neff, A. W.

1984-01-01

The development of animal systems is described in terms of a series of overlapping phases: pattern specification; differentiation; growth; and aging. The extent to which altered (micro) gravity (g) affects those phases is briefly reviewed for several animal systems. As a model, amphibian egg/early embryo is described. Recent data derived from clinostat protocols indicates that microgravity simulation alters early pattern specification (dorsal/ventral polarity) but does not adversely influence subsequent morphogenesis. Possible explanations for the absence of catastrophic microgravity effects on amphibian embryogenesis are discussed.

Germ cell specification and ovary structure in the rotifer Brachionus plicatilis.

PubMed

Smith, James M; Cridge, Andrew G; Dearden, Peter K

2010-08-02

The segregation of the germline from somatic tissues is an essential process in the development of all animals. Specification of the primordial germ cells (PGCs) takes place via different strategies across animal phyla; either specified early in embryogenesis by the inheritance of maternal determinants in the cytoplasm of the oocyte ('preformation') or selected later in embryonic development from undifferentiated precursors by a localized inductive signal ('epigenesis'). Here we investigate the specification and development of the germ cells in the rotifer Brachionus plicatilis, a member of the poorly-characterized superphyla Lophotrochozoa, by isolating the Brachionus homologues of the conserved germ cell markers vasa and nanos, and examining their expression using in situ hybridization. Bpvasa and Bpnos RNA expression have very similar distributions in the Brachionus ovary, showing ubiquitous expression in the vitellarium, with higher levels in the putative germ cell cluster. Bpvas RNA expression is present in freshly laid eggs, remaining ubiquitous in embryos until at least the 96 cell stage after which expression narrows to a small cluster of cells at the putative posterior of the embryo, consistent with the developing ovary. Bpnos RNA expression is also present in just-laid eggs but expression is much reduced by the four-cell stage and absent by the 16-cell stage. Shortly before hatching of the juvenile rotifer from the egg, Bpnos RNA expression is re-activated, located in a subset of posterior cells similar to those expressing Bpvas at the same stage. The observed expression of vasa and nanos in the developing B. plicatilis embryo implies an epigenetic origin of primordial germ cells in Rotifer.

Identification of early zygotic genes in the yellow fever mosquito Aedes aegypti and discovery of a motif involved in early zygotic genome activation.

PubMed

Biedler, James K; Hu, Wanqi; Tae, Hongseok; Tu, Zhijian

2012-01-01

During early embryogenesis the zygotic genome is transcriptionally silent and all mRNAs present are of maternal origin. The maternal-zygotic transition marks the time over which embryogenesis changes its dependence from maternal RNAs to zygotically transcribed RNAs. Here we present the first systematic investigation of early zygotic genes (EZGs) in a mosquito species and focus on genes involved in the onset of transcription during 2-4 hr. We used transcriptome sequencing to identify the "pure" (without maternal expression) EZGs by analyzing transcripts from four embryonic time ranges of 0-2, 2-4, 4-8, and 8-12 hr, which includes the time of cellular blastoderm formation and up to the start of gastrulation. Blast of 16,789 annotated transcripts vs. the transcriptome reads revealed evidence for 63 (P<0.001) and 143 (P<0.05) nonmaternally derived transcripts having a significant increase in expression at 2-4 hr. One third of the 63 EZG transcripts do not have predicted introns compared to 10% of all Ae. aegypti genes. We have confirmed by RT-PCR that zygotic transcription starts as early as 2-3 hours. A degenerate motif VBRGGTA was found to be overrepresented in the upstream sequences of the identified EZGs using a motif identification software called SCOPE. We find evidence for homology between this motif and the TAGteam motif found in Drosophila that has been implicated in EZG activation. A 38 bp sequence in the proximal upstream sequence of a kinesin light chain EZG (KLC2.1) contains two copies of the mosquito motif. This sequence was shown to support EZG transcription by luciferase reporter assays performed on injected early embryos, and confers early zygotic activity to a heterologous promoter from a divergent mosquito species. The results of these studies are consistent with the model of early zygotic genome activation via transcriptional activators, similar to what has been found recently in Drosophila.

Donor cell differentiation, reprogramming, and cloning efficiency: elusive or illusive correlation?

PubMed

Oback, B; Wells, D N

2007-05-01

Compared to other assisted reproductive technologies, mammalian nuclear transfer (NT) cloning is inefficient in generating viable offspring. It has been postulated that nuclear reprogramming and cloning efficiency can be increased by choosing less differentiated cell types as nuclear donors. This hypothesis is mainly supported by comparative mouse cloning experiments using early blastomeres, embryonic stem (ES) cells, and terminally differentiated somatic donor cells. We have re-evaluated these comparisons, taking into account different NT procedures, the use of donor cells from different genetic backgrounds, sex, cell cycle stages, and the lack of robust statistical significance when post-blastocyst development is compared. We argue that while the reprogrammability of early blastomeres appears to be much higher than that of somatic cells, it has so far not been conclusively determined whether differentiation status affects cloning efficiency within somatic donor cell lineages. Copyright (c) 2006 Wiley-Liss, Inc.

The large Maf factor Traffic Jam controls gonad morphogenesis in Drosophila.

PubMed

Li, Michelle A; Alls, Jeffrey D; Avancini, Rita M; Koo, Karen; Godt, Dorothea

2003-11-01

Interactions between somatic and germline cells are critical for the normal development of egg and sperm. Here we show that the gene traffic jam (tj) produces a soma-specific factor that controls gonad morphogenesis and is required for female and male fertility. tj encodes the only large Maf factor in Drosophila melanogaster, an orthologue of the atypical basic Leu zipper transcription factors c-Maf and MafB/Kreisler in vertebrates. Expression of tj occurs in somatic gonadal cells that are in direct contact with germline cells throughout development. In tj mutant gonads, somatic cells fail to inter-mingle and properly envelop germline cells, causing an early block in germ cell differentiation. In addition, tj mutant somatic cells show an increase in the level of expression for several adhesion molecules. We propose that tj is a critical modulator of the adhesive properties of somatic cells, facilitating germline-soma interactions that are essential for germ cell differentiation.

Epilepsy as a systemic condition: Link with somatic comorbidities.

PubMed

Novy, J; Bell, G S; Peacock, J L; Sisodiya, S M; Sander, J W

2017-10-01

People with epilepsy have more concomitant medical conditions than the general population; these comorbidities play an important role in premature mortality. We sought to generate explanatory hypotheses about the co-occurrence of somatic comorbidities and epilepsy, avoiding causal and treatment-resultant biases. We collected clinical, demographic and somatic comorbidity data for 2016 consecutive adults with epilepsy undergoing assessment at a tertiary centre and in 1278 people with epilepsy in the community. Underlying causes of epilepsy were not classed as comorbidities. Somatic comorbidities were more frequent in the referral centre (49%) where people more frequently had active epilepsy than in the community (36%). Consistent risk factors for comorbidities were found in both cohorts. Using multivariable ordinal regression adjusted for age, longer epilepsy duration and an underlying brain lesion were independently associated with a smaller burden of somatic conditions. The treatment burden, measured by the number of drugs to which people were exposed, was not an independent predictor. Shorter epilepsy duration was a predictor for conditions that conceivably harbour significant mortality risks. Somatic comorbidities do not occur randomly in relation to epilepsy; having more severe epilepsy seems to be a risk factor. Independently from age, the early period after epilepsy onset appears to be at particular risk, although it is not clear whether this relates to an early mortality or to a later decrease in the burden of comorbidities. These results suggest that, for some people, epilepsy should be considered a systemic condition not limited to the CNS. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Innexin2 gap junctions in somatic support cells are required for cyst formation and for egg chamber formation in Drosophila.

PubMed

Mukai, Masanori; Kato, Hirotaka; Hira, Seiji; Nakamura, Katsuhiro; Kita, Hiroaki; Kobayashi, Satoru

2011-01-01

Germ cells require intimate associations with surrounding somatic cells during gametogenesis. During oogenesis, gap junctions mediate communication between germ cells and somatic support cells. However, the molecular mechanisms by which gap junctions regulate the developmental processes during oogenesis are poorly understood. We have identified a female sterile allele of innexin2 (inx2), which encodes a gap junction protein in Drosophila. In females bearing this inx2 allele, cyst formation and egg chamber formation are impaired. In wild-type germaria, Inx2 is strongly expressed in escort cells and follicle cells, both of which make close contact with germline cells. We show that inx2 function in germarial somatic cells is required for the survival of early germ cells and promotes cyst formation, probably downstream of EGFR pathway, and that inx2 function in follicle cells promotes egg chamber formation through the regulation of DE-cadherin and Bazooka (Baz) at the boundary between germ cells and follicle cells. Furthermore, genetic experiments demonstrate that inx2 interacts with the zero population growth (zpg) gene, which encodes a germline-specific gap junction protein. These results indicate a multifunctional role for Inx2 gap junctions in somatic support cells in the regulation of early germ cell survival, cyst formation and egg chamber formation. Inx2 gap junctions may mediate the transfer of nutrients and signal molecules between germ cells and somatic support cells, as well as play a role in the regulation of cell adhesion. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

[The hardest battles begin after the war].

PubMed

Sodemann, Morten; Svabo, Arndis; Jacobsen, Arne

2010-01-11

While psychic effects of war trauma are well-described, the somatic long-term consequences of war trauma have not previously been described. In three clinical cases from the Migrant Health Clinic at Odense University Hospital, we describe the complicated somatic problems which can be associated with a refugee status. The cross disciplinary team chose three cases that describe the long-term effects of war trauma. Post-traumatic stress disorder (PTSD) can appear 10-20 years after a primary war-related trauma and secondary trauma after the arrival in Denmark trigger and prolong post-traumatic stress symptoms with a range of somatic symptoms. Warning signs of an underlying PTSD disorder have often been present for many years, but overlooked or ignored. Many patients with PTSD and somatic symptoms loose previously acquired language skills, disintegrate and drop out of the labour market after 3-4 years in Denmark. Somatic symptoms along with PTSD can develop into a seriously complicated condition that requires skilled cross-disciplinary management. Experience from the Cross Disciplinary Migrant Health Clinic shows that by investing time in obtaining a full clinical and social history it is possible to increase the quality of life of these patients. Early screening and early specialized cross disciplinary and cross sectorial management are crucial to secure and maintain integration, but unfortunately the long waiting list to institutions that treat PTSD contributes to the high level of disintegration.

Controlling the Messenger: Regulated Translation of Maternal mRNAs in Xenopus laevis Development

PubMed Central

Fox, Catherine A.; Dowdle, Megan E.; Blaser, Susanne Imboden; Chung, Andy; Park, Sookhee

2017-01-01

The selective translation of maternal mRNAs encoding cell-fate determinants drives the earliest decisions of embryogenesis that establish the vertebrate body plan. This chapter will discuss studies in Xenopus laevis that provide insights into mechanisms underlying this translational control. Xenopus has been a powerful model organism for many discoveries relevant to the translational control of maternal mRNAs because of the large size of its oocytes and eggs that allow for microinjection of molecules and the relative ease of manipulating the oocyte to egg transition (maturation) and fertilization in culture. Consequently, many key studies have focused on the expression of maternal mRNAs during the oocyte to egg transition (the meiotic cell cycle) and the rapid cell divisions immediately following fertilization. This research has made seminal contributions to our understanding of translational regulatory mechanisms, but while some of the mRNAs under consideration at these stages encode cell-fate determinants, many encode cell cycle regulatory proteins that drive these early cell cycles. In contrast, while maternal mRNAs encoding key developmental (i.e., cell-fate) regulators that function after the first cleavage stages may exploit aspects of these foundational mechanisms, studies reveal that these mRNAs must also rely on distinct and, as of yet, incompletely understood mechanisms. These findings are logical because the functions of such developmental regulatory proteins have requirements distinct from cell cycle regulators, including becoming relevant only after fertilization and then only in specific cells of the embryo. Indeed, key maternal cell-fate determinants must be made available in exquisitely precise amounts (usually low), only at specific times and in specific cells during embryogenesis. To provide an appreciation for the regulation of maternal cell-fate determinant expression, an overview of the maternal phase of Xenopus embryogenesis will be presented. This section will be followed by a review of translational mechanisms operating in oocytes, eggs, and early cleavage-stage embryos and conclude with a discussion of how the regulation of key maternal cell-fate determinants at the level of translation functions in Xenopus embryogenesis. A key theme is that the molecular asymmetries critical for forming the body axes are established and further elaborated upon by the selective temporal and spatial regulation of maternal mRNA translation. PMID:27975270

Effect of PMA-induced protein kinase C activation on development and apoptosis in early zebrafish embryos.

PubMed

Hrubik, Jelena; Glisic, Branka; Samardzija, Dragana; Stanic, Bojana; Pogrmic-Majkic, Kristina; Fa, Svetlana; Andric, Nebojsa

2016-12-01

Protein kinase C (PKC) isoforms have been implicated in several key steps during early development, but the consequences of xenobiotic-induced PKC activation during early embryogenesis are still unknown. In this study, zebrafish embryos were exposed to a range of phorbol 12-myristate 13-acetate (PMA) concentrations (0-200μg/L) at different time points after fertilization. Results showed that 200μgPMA/L caused development of yolk bags, cardiac edema, slow blood flow, pulsating blood flow, slow pulse, elongated heart, lack of tail fins, curved tail, and coagulation. PMA exposure decreased survival rate of the embryos starting within the first 24h and becoming more pronounced after prolonged exposure (96h). PMA increased the number of apoptotic cells in the brain region as demonstrated by acridine orange staining and caused up-regulation of caspase 9 (casp9) and p53 up-regulated modulator of apoptosis (puma) mRNA in whole embryos. PMA caused oxidative stress in the embryos as demonstrated by decreased mRNA expression of catalase and superoxide dismutase 2. Inhibition of Pkc with GF109203X improved overall survival rate, reduced apoptosis in the brain and decreased expression of casp9 and puma in the PMA-exposed embryos. However, Pkc inhibition neither prevented development of deformities nor reversed oxidative stress in the PMA-exposed embryos. These data suggest that direct over-activation of Pkc during early embryogenesis of zebrafish is associated with apoptosis and decreased survival rate of the embryos. Copyright © 2016 Elsevier Inc. All rights reserved.

An RGDS peptide-binding receptor, FR-1R, localizes to the basal side of the ectoderm and to primary mesenchyme cells in sand dollar embryos.

PubMed

Katow, H; Sofuku, S

2001-10-01

Immunoblotting using polyclonal antibodies (pAb) raised against an FR-1 receptor (FR-1R), a 57 kDa Arg-Gly-Asp-Ser (RGDS)-binding protein, of the sand dollar Clypeaster japonicus showed that the pAb monospecifically bound to the protein. FR-1R was present in purified plasma membrane, suggesting that the protein is a membrane-bound protein. The molecular structure of FR-1R did not change throughout the early embryogenesis, whereas its expression changed significantly during this period. FR-1R was present in the cortex of unfertilized eggs and was then transferred to the hyaline layer soon after the fertilization. The hyaline layer retained FR-1R immunoreactivity during early embryogenesis. FR-1R appeared on the basal side of the ectoderm at the morula stage and was retained basolaterally, at least, to the early gastrula stage. In mesenchyme blastulae, FR-1R was also present on the surface of primary mesenchyme cells (PMC). FR-1R was localized on the basal side of the ectoderm in early gastrulae, exclusively at the place where PMC formed ventrolateral aggregates, and at the apical tuft ectoderm. In vitro, PMC bound to FR-1R and its binding was inhibited in the presence of a synthetic RGDS peptide or the pAb. The pAb introduced into the blastocoele perturbed PMC migration and gastrulation. FR-1R was weakly recognized by antihuman integrin beta5 subunit pAb.

Survival of ovarian somatic cells during sex change in the protogynous wrasse, Halichoeres trimaculatus.

PubMed

Nozu, Ryo; Horiguchi, Ryo; Murata, Ryosuke; Kobayashi, Yasuhisa; Nakamura, Masaru

2013-02-01

The three-spot wrasse (Halichoeres trimaculatus), which inhabits the coral reefs of Okinawa, changes sex from female to male. Sex change in this species is controlled by a social system. Oocytes disappear completely from the ovary, and male germ cells and somatic cells comprising testicular tissue arise a new during the sex change process. However, little is known of the fate and origin of the gonadal tissue-forming cells during sex change. In particular, the fate of ovarian somatic cells has not been determined, although the ovarian tissue regresses histologically. To approach this question, we analyzed apoptosis and cell proliferation in the sex-changing gonads. Unexpectedly, we found that few apoptotic somatic cells were present during sex change, suggesting that ovarian somatic cells might survive during the regression of the ovarian tissue. On the other hand, cell proliferation was detected in many granulosa cells surrounding the degenerating oocytes, a few epithelial cells covering ovigerous lamella and a few somatic cells associated with gonial germ cells at an early stage of sex change. Then, we found that proliferative ovarian somatic cells remained in the gonads late in the sex change process. Based on these results, we concluded that some functional somatic cells of the ovary are reused as testicular somatic cells during the gonadal sex change in the three-spot wrasse.

Micropropagation of African violet (Saintpaulia ionantha Wendl.).

PubMed

Shukla, Mukund; Sullivan, J Alan; Jain, Shri Mohan; Murch, Susan J; Saxena, Praveen K

2013-01-01

Micropropagation is an important tool for rapid multiplication and the creation of genetic variability in African violets (Saintpaulia ionantha Wendl.). Successful in vitro propagation depends on the specific requirements and precise manipulation of various factors such as the type of explants used, physiological state of the mother plant, plant growth regulators in the culture medium, and growth conditions. Development of cost-effective protocols with a high rate of multiplication is a crucial requirement for commercial application of micropropagation. The current chapter describes an optimized protocol for micropropagation of African violets using leaf explants obtained from in vitro grown plants. In this process, plant regeneration occurs via both somatic embryogenesis and shoot organogenesis simultaneously in the explants induced with the growth regulator thidiazuron (TDZ; N-phenyl-N'-1,2,3-thidiazol-5-ylurea). The protocol is simple, rapid, and efficient for large-scale propagation of African violet and the dual routes of regeneration allow for multiple applications of the technology from simple clonal propagation to induction or selection of variants to the production of synthetic seeds.

Copy Number Variation Is a Fundamental Aspect of the Placental Genome

PubMed Central

Hannibal, Roberta L.; Chuong, Edward B.; Rivera-Mulia, Juan Carlos; Gilbert, David M.; Valouev, Anton; Baker, Julie C.

2014-01-01

Discovery of lineage-specific somatic copy number variation (CNV) in mammals has led to debate over whether CNVs are mutations that propagate disease or whether they are a normal, and even essential, aspect of cell biology. We show that 1,000N polyploid trophoblast giant cells (TGCs) of the mouse placenta contain 47 regions, totaling 138 Megabases, where genomic copies are underrepresented (UR). UR domains originate from a subset of late-replicating heterochromatic regions containing gene deserts and genes involved in cell adhesion and neurogenesis. While lineage-specific CNVs have been identified in mammalian cells, classically in the immune system where V(D)J recombination occurs, we demonstrate that CNVs form during gestation in the placenta by an underreplication mechanism, not by recombination nor deletion. Our results reveal that large scale CNVs are a normal feature of the mammalian placental genome, which are regulated systematically during embryogenesis and are propagated by a mechanism of underreplication. PMID:24785991

DNA methylation restricts lineage-specific functions of transcription factor Gata4 during embryonic stem cell differentiation.

PubMed

Oda, Masaaki; Kumaki, Yuichi; Shigeta, Masaki; Jakt, Lars Martin; Matsuoka, Chisa; Yamagiwa, Akiko; Niwa, Hitoshi; Okano, Masaki

2013-06-01

DNA methylation changes dynamically during development and is essential for embryogenesis in mammals. However, how DNA methylation affects developmental gene expression and cell differentiation remains elusive. During embryogenesis, many key transcription factors are used repeatedly, triggering different outcomes depending on the cell type and developmental stage. Here, we report that DNA methylation modulates transcription-factor output in the context of cell differentiation. Using a drug-inducible Gata4 system and a mouse embryonic stem (ES) cell model of mesoderm differentiation, we examined the cellular response to Gata4 in ES and mesoderm cells. The activation of Gata4 in ES cells is known to drive their differentiation to endoderm. We show that the differentiation of wild-type ES cells into mesoderm blocks their Gata4-induced endoderm differentiation, while mesoderm cells derived from ES cells that are deficient in the DNA methyltransferases Dnmt3a and Dnmt3b can retain their response to Gata4, allowing lineage conversion from mesoderm cells to endoderm. Transcriptome analysis of the cells' response to Gata4 over time revealed groups of endoderm and mesoderm developmental genes whose expression was induced by Gata4 only when DNA methylation was lost, suggesting that DNA methylation restricts the ability of these genes to respond to Gata4, rather than controlling their transcription per se. Gata4-binding-site profiles and DNA methylation analyses suggested that DNA methylation modulates the Gata4 response through diverse mechanisms. Our data indicate that epigenetic regulation by DNA methylation functions as a heritable safeguard to prevent transcription factors from activating inappropriate downstream genes, thereby contributing to the restriction of the differentiation potential of somatic cells.

Spatial localization of EGF family ligands and receptors in the zebrafish ovarian follicle and their expression profiles during folliculogenesis.

PubMed

Tse, Anna Chung-Kwan; Ge, Wei

2010-07-01

The roles of epidermal growth factor (EGF) family in the ovary have received increasing attention recently. Despite this, the production sites of EGF family members in the ovarian follicle still remain controversial. Using zebrafish as the model, the present study investigated spatial distribution of several EGF family ligands and receptors in the follicle as well as their temporal expression profiles during folliculogenesis. RT-PCR analysis on the somatic follicle layer and oocyte revealed that all EGF family ligands examined (egf, tgfa, btc and hbegf) were mostly or exclusively expressed in the oocyte. In contrast, their common receptor (egfr) was expressed exclusively in the follicle layer. By comparison, members of activin family showed an opposite pattern of distribution. Activin subunits (inhbaa and inhbb) were both expressed exclusively in the follicle layer whereas activin receptors and follistatin were abundantly present in the oocyte. During folliculogenesis, egf, tgfa and hbegf increased their expression together with egfr in the fast secondary growth phase. The developmental profiles of EGF family during embryogenesis appeared to argue for an important role for EGF family in folliculogenesis rather than embryogenesis as maternal molecules. The present study provided clear evidence for the existence of two paracrine pathways in the follicle, the oocyte-derived EGF family ligands and follicle cell-derived activins, which may mediate oocyte-to-follicle cell and follicle cell-to-oocyte communications, respectively. The functional relationship between these two signaling systems in the follicle is suggested by the observation that all four EGFR ligands examined significantly stimulated activin subunit expression in cultured follicle cells. (c) 2009 Elsevier Inc. All rights reserved.

Effects of biotic and abiotic factors on the oxygen content of green sea turtle nests during embryogenesis.

PubMed

Chen, Chiu-Lin; Wang, Chun-Chun; Cheng, I-Jiunn

2010-10-01

Several biotic and abiotic factors can influence nest oxygen content during embryogenesis. Several of these factors were determined during each developmental stage of green sea turtle embryos on Wan-an Island, Penghu Archipelago, Taiwan. We examined oxygen content in 7 nests in 2007 and 11 in 2008. Oxygen in the adjacent sand, total and viable clutch sizes, air, sand and nest temperatures, and sand characters of each nest were also determined. Oxygen content was lower in late stages than in the early and middle stages. It was also lower in the middle layer than in the upper and bottom layers. Nest temperature showed opposite trends, reaching its maximum value in late stages of development. Nest oxygen content was influenced by fraction of viable eggs, total clutch sizes, sand temperatures, maximum nest temperature and maximum change in the nest temperature during incubation. Clutch size during embryogenesis was the most influential factor overall. However, the major influential factors were different for different developmental stages. In the first half of the incubation, the development rate was low, and the change in the nest oxygen content was influenced mainly by the clutch size. During the second half, the rapid embryonic development rate became the dominant factor, and hatchling activities caused even greater oxygen consumption during the last stage of development.

Cell dedifferentiation, callus induction and somatic embryogenesis in Crataegus spp.

PubMed

Taimori, N; Kahrizi, D; Abdossi, V; Papzan, A H

2016-09-30

The present study describes the effects of light conditions, different kinds and concentrations of auxins [Naphthylacetic acid (NAA) and dichlorophenoxyacetic acid (2,4-D)] with cytokinin (Kin) in MS medium on callus induction and embryogenesis in Crataegus pseudoheterophylla, C. aronia and C.meyeri. At first leave explants sections were cultured on different combinations of plant growth regulators in dark and light for callus initiation and light conditions to evaluation the percentage and duration of survival, callus diameter, callus fresh weight and dry. Results of effects of plant growth regulators and light conditions on callus initiation revealed that highest percentage of callus initiation leaves in treatment (0.5 mg/l 2.4-D+0.5 mg/l KIN) for species C.pseudoheterophylla in dark conditions (100%). Dark conditions (100%) were more effective on callogenesis than light conditions (Photoperiodicity of 16-h and at light intensity of 40 µmol m-2 s-1). The callus induction of in vitro (64-100%) leaves was better than the ex vitro ones (0-100%). The combination of 2,4-D and Kin of in vitro leaves callogenesis has been indicated faster (one weeks) than the other combinations. The results also showed that the highest percentage (100%) and survival duration (6 months) was found in species C. pseudoheterophylla and C. meyeri in 0.1 mg/l 2,4.D + 0.5 mg/l KIN and 0.5 mg/l 2,4.D + 0.5 mg/l Kin. The minimum survival (0%) was absorbed in species C. aronia in 1 mg/l NAA. Maximum callus (10.63 and 10.00 mm respectively) was shown in 0.1 mg/l 2,4.D + 0.5 mg/l Kin and 0.5 mg/l 2,4.D + 0.5 mg/l Kin and was not significant differences after five week among species. The results showed that the highest fresh (1081.49 mg) and dry weight (506.88 and 506.98 mg respectively) was absorbed in species C. pseudoheterophylla in 0.1 mg/l 2,4.D + 0.5 mg/l Kin and 0.5 mg/l 2,4.D + 0.5 mg/l Kin. The embryogenesis was not occurred in any plant growth regulator combinations and species. The results of this study suggested that using 2,4-D with cytokinin (Kin) would be more beneficial for callogenesis.

Drosophila melanogaster as a model system for assessing development under conditions of microgravity

NASA Technical Reports Server (NTRS)

Abbott, M. K.; Hilgenfeld, R. B.; Denell, R. E.; Spooner, B. S. (Principal Investigator)

1992-01-01

More is known about the regulation of early developmental events in Drosophila than any other animal. In addition, its size and short life cycle make it a facile experimental system. Since developmental perturbations have been demonstrated when both oogenesis and embryogenesis occur in the space environment, there is a strong rationale for using this organism for the elucidation of specific gravity-sensitive developmental events.

DNA methyltransferases and stress-related genes expression in zebrafish larvae after exposure to heat and copper during reprogramming of DNA methylation.

PubMed

Dorts, Jennifer; Falisse, Elodie; Schoofs, Emilie; Flamion, Enora; Kestemont, Patrick; Silvestre, Frédéric

2016-10-12

DNA methylation, a well-studied epigenetic mark, is important for gene regulation in adulthood and for development. Using genetic and epigenetic approaches, the present study aimed at evaluating the effects of heat stress and copper exposure during zebrafish early embryogenesis when patterns of DNA methylation are being established, a process called reprogramming. Embryos were exposed to 325 μg Cu/L from fertilization (<1 h post fertilization - hpf) to 4 hpf at either 26.5 °C or 34 °C, followed by incubation in clean water at 26.5 °C till 96 hpf. Significant increased mortality rates and delayed hatching were observed following exposure to combined high temperature and Cu. Secondly, both stressors, alone or in combination, significantly upregulated the expression of de novo DNA methyltransferase genes (dnmt3) along with no differences in global cytosine methylation level. Finally, Cu exposure significantly increased the expression of metallothionein (mt2) and heat shock protein (hsp70), the latter being also increased following exposure to high temperature. These results highlighted the sensitivity of early embryogenesis and more precisely of the reprogramming period to environmental challenges, in a realistic situation of combined stressors.

Acid stress mediated adaptive divergence in ion channel function during embryogenesis in Rana arvalis

PubMed Central

Shu, Longfei; Laurila, Anssi; Räsänen, Katja

2015-01-01

Ion channels and pumps are responsible for ion flux in cells, and are key mechanisms mediating cellular function. Many environmental stressors, such as salinity and acidification, are known to severely disrupt ionic balance of organisms thereby challenging fitness of natural populations. Although ion channels can have several vital functions during early life-stages (e.g. embryogenesis), it is currently not known i) how developing embryos maintain proper intracellular conditions when exposed to environmental stress and ii) to what extent environmental stress can drive intra-specific divergence in ion channels. Here we studied the moor frog, Rana arvalis, from three divergent populations to investigate the role of different ion channels and pumps for embryonic survival under acid stress (pH 4 vs 7.5) and whether populations adapted to contrasting acidities differ in the relative role of different ion channel/pumps. We found that ion channels that mediate Ca2+ influx are essential for embryonic survival under acidic pH, and, intriguingly, that populations differ in calcium channel function. Our results suggest that adaptive divergence in embryonic acid stress tolerance of amphibians may in part be mediated by Ca2+ balance. We suggest that ion flux may mediate adaptive divergence of natural populations at early life-stages in the face of environmental stress. PMID:26381453

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

Predictors of somatic symptoms: a five year follow up of adolescents

PubMed Central

Poikolainen, K; Aalto-Setala, T; Marttunen, M; Tuulio-Henriksson, A; Lonnqvist, J

2000-01-01

BACKGROUND—Somatisation is common among adolescents.
AIMS—To study factors predicting somatisation later in adulthood.
METHODS—Self report questionnaires were administered at baseline examination in 1990 to students (mean age 16.8 years) in schools, and by mail five years later. Results are based on the 615 subjects with no serious disease or injury at baseline.
RESULTS—Regression analyses showed that in men the level of somatic symptoms in 1995 was significantly predicted by the respective level in 1990 and by relief smoking. In women, the level of somatic symptoms in 1995 was significantly predicted by the respective level in 1990, self esteem, and the number of negative life events in 1990. After exclusion of cases with a long standing disease in 1995, the multivariate results remained materially similar except that self esteem was no longer significant among women.
CONCLUSION—These findings may help in early identification of adolescents with somatisation persisting into early adulthood.

 PMID:11040143

Distinct cellular pathways select germline-encoded and somatically mutated antibodies into immunological memory

PubMed Central

Kaji, Tomohiro; Ishige, Akiko; Hikida, Masaki; Taka, Junko; Hijikata, Atsushi; Kubo, Masato; Nagashima, Takeshi; Takahashi, Yoshimasa; Kurosaki, Tomohiro; Okada, Mariko; Ohara, Osamu

2012-01-01

One component of memory in the antibody system is long-lived memory B cells selected for the expression of somatically mutated, high-affinity antibodies in the T cell–dependent germinal center (GC) reaction. A puzzling observation has been that the memory B cell compartment also contains cells expressing unmutated, low-affinity antibodies. Using conditional Bcl6 ablation, we demonstrate that these cells are generated through proliferative expansion early after immunization in a T cell–dependent but GC-independent manner. They soon become resting and long-lived and display a novel distinct gene expression signature which distinguishes memory B cells from other classes of B cells. GC-independent memory B cells are later joined by somatically mutated GC descendants at roughly equal proportions and these two types of memory cells efficiently generate adoptive secondary antibody responses. Deletion of T follicular helper (Tfh) cells significantly reduces the generation of mutated, but not unmutated, memory cells early on in the response. Thus, B cell memory is generated along two fundamentally distinct cellular differentiation pathways. One pathway is dedicated to the generation of high-affinity somatic antibody mutants, whereas the other preserves germ line antibody specificities and may prepare the organism for rapid responses to antigenic variants of the invading pathogen. PMID:23027924

Mitochondrial DNA transmission and confounding mitochondrial influences in cloned cattle and pigs.

PubMed

Takeda, Kumiko

2013-04-01

Although somatic cell nuclear transfer (SCNT) is a powerful tool for production of cloned animals, SCNT embryos generally have low developmental competency and many abnormalities. The interaction between the donor nucleus and the enucleated ooplasm plays an important role in early embryonic development, but the underlying mechanisms that negatively impact developmental competency remain unclear. Mitochondria have a broad range of critical functions in cellular energy supply, cell signaling, and programmed cell death; thus, affect embryonic and fetal development. This review focuses on mitochondrial considerations influencing SCNT techniques in farm animals. Donor somatic cell mitochondrial DNA (mtDNA) can be transmitted through what has been considered a "bottleneck" in mitochondrial genetics via the SCNT maternal lineage. This indicates that donor somatic cell mitochondria have a role in the reconstructed cytoplasm. However, foreign somatic cell mitochondria may affect the early development of SCNT embryos. Nuclear-mitochondrial interactions in interspecies/intergeneric SCNT (iSCNT) result in severe problems. A major biological selective pressure exists against survival of exogenous mtDNA in iSCNT. Yet, mtDNA differences in SCNT animals did not reflect transfer of proteomic components following proteomic analysis. Further study of nuclear-cytoplasmic interactions is needed to illuminate key developmental characteristics of SCNT animals associated with mitochondrial biology.

Argonaute-1 functions as a mitotic regulator by controlling Cyclin B during Drosophila early embryogenesis.

PubMed

Pushpavalli, Sreerangam N C V L; Sarkar, Arpita; Bag, Indira; Hunt, Clayton R; Ramaiah, M Janaki; Pandita, Tej K; Bhadra, Utpal; Pal-Bhadra, Manika

2014-02-01

The role of Ago-1 in microRNA (miRNA) biogenesis has been thoroughly studied, but little is known about its involvement in mitotic cell cycle progression. In this study, we established evidence of the regulatory role of Ago-1 in cell cycle control in association with the G2/M cyclin, cyclin B. Immunostaining of early embryos revealed that the maternal effect gene Ago-1 is essential for proper chromosome segregation, mitotic cell division, and spindle fiber assembly during early embryonic development. Ago-1 mutation resulted in the up-regulation of cyclin B-Cdk1 activity and down-regulation of p53, grp, mei-41, and wee1. The increased expression of cyclin B in Ago-1 mutants caused less stable microtubules and probably does not produce enough force to push the nuclei to the cortex, resulting in a decreased number of pole cells. The role of cyclin B in mitotic defects was further confirmed by suppressing the defects in the presence of one mutant copy of cyclin B. We identified involvement of 2 novel embryonic miRNAs--miR-981 and miR--317-for spatiotemporal regulation of cyclin B. In summary, our results demonstrate that the haploinsufficiency of maternal Ago-1 disrupts mitotic chromosome segregation and spindle fiber assembly via miRNA-guided control during early embryogenesis in Drosophila. The increased expression of cyclin B-Cdk1 and decreased activity of the Cdk1 inhibitor and cell cycle checkpoint proteins (mei-41 and grp) in Ago-1 mutant embryos allow the nuclei to enter into mitosis prematurely, even before completion of DNA replication. Thus, our results have established a novel role of Ago-1 as a regulator of the cell cycle.

Lysophosphatidic acid acts as a nutrient-derived developmental cue to regulate early hematopoiesis

PubMed Central

Li, Haisen; Yue, Rui; Wei, Bin; Gao, Ge; Du, Jiulin; Pei, Gang

2014-01-01

Primitive hematopoiesis occurs in the yolk sac blood islands during vertebrate embryogenesis, where abundant phosphatidylcholines (PC) are available as important nutrients for the developing embryo. However, whether these phospholipids also generate developmental cues to promote hematopoiesis is largely unknown. Here, we show that lysophosphatidic acid (LPA), a signaling molecule derived from PC, regulated hemangioblast formation and primitive hematopoiesis. Pharmacological and genetic blockage of LPA receptor 1 (LPAR1) or autotoxin (ATX), a secretory lysophospholipase that catalyzes LPA production, inhibited hematopoietic differentiation of mouse embryonic stem cells and impaired the formation of hemangioblasts. Mechanistic experiments revealed that the regulatory effect of ATX-LPA signaling was mediated by PI3K/Akt-Smad pathway. Furthermore, during in vivo embryogenesis in zebrafish, LPA functioned as a developmental cue for hemangioblast formation and primitive hematopoiesis. Taken together, we identified LPA as an important nutrient-derived developmental cue for primitive hematopoiesis as well as a novel mechanism of hemangioblast regulation. PMID:24829209

Epigenetic regulation of left-right asymmetry by DNA methylation.

PubMed

Wang, Lu; Liu, Zhibin; Lin, Hao; Ma, Dongyuan; Tao, Qinghua; Liu, Feng

2017-10-16

DNA methylation is a major epigenetic modification; however, the precise role of DNA methylation in vertebrate development is still not fully understood. Here, we show that DNA methylation is essential for the establishment of the left-right (LR) asymmetric body plan during vertebrate embryogenesis. Perturbation of DNA methylation by depletion of DNA methyltransferase 1 ( dnmt1 ) or dnmt3bb.1 in zebrafish embryos leads to defects in dorsal forerunner cell (DFC) specification or collective migration, laterality organ malformation, and disruption of LR patterning. Knockdown of dnmt1 in Xenopus embryos also causes similar defects. Mechanistically, loss of dnmt1 function induces hypomethylation of the lefty2 gene enhancer and promotes lefty2 expression, which consequently represses Nodal signaling in zebrafish embryos. We also show that Dnmt3bb.1 regulates collective DFC migration through cadherin 1 (Cdh1). Taken together, our data uncover dynamic DNA methylation as an epigenetic mechanism to control LR determination during early embryogenesis in vertebrates. © 2017 The Authors.

Lysophosphatidic acid acts as a nutrient-derived developmental cue to regulate early hematopoiesis.

PubMed

Li, Haisen; Yue, Rui; Wei, Bin; Gao, Ge; Du, Jiulin; Pei, Gang

2014-06-17

Primitive hematopoiesis occurs in the yolk sac blood islands during vertebrate embryogenesis, where abundant phosphatidylcholines (PC) are available as important nutrients for the developing embryo. However, whether these phospholipids also generate developmental cues to promote hematopoiesis is largely unknown. Here, we show that lysophosphatidic acid (LPA), a signaling molecule derived from PC, regulated hemangioblast formation and primitive hematopoiesis. Pharmacological and genetic blockage of LPA receptor 1 (LPAR1) or autotoxin (ATX), a secretory lysophospholipase that catalyzes LPA production, inhibited hematopoietic differentiation of mouse embryonic stem cells and impaired the formation of hemangioblasts. Mechanistic experiments revealed that the regulatory effect of ATX-LPA signaling was mediated by PI3K/Akt-Smad pathway. Furthermore, during in vivo embryogenesis in zebrafish, LPA functioned as a developmental cue for hemangioblast formation and primitive hematopoiesis. Taken together, we identified LPA as an important nutrient-derived developmental cue for primitive hematopoiesis as well as a novel mechanism of hemangioblast regulation. © 2014 The Authors.

Improved Resistance to Controlled Deterioration in Transgenic Seeds1[W][OA

PubMed Central

Prieto-Dapena, Pilar; Castaño, Raúl; Almoguera, Concepción; Jordano, Juan

2006-01-01

We show that seed-specific overexpression of the sunflower (Helianthus annuus) HaHSFA9 heat stress transcription factor (HSF) in tobacco (Nicotiana tabacum) enhances the accumulation of heat shock proteins (HSPs). Among these proteins were HSP101 and a subset of the small HSPs, including proteins that accumulate only during embryogenesis in the absence of thermal stress. Levels of late embryogenesis abundant proteins or seed oligosaccharides, however, were not affected. In the transgenic seeds, a high basal thermotolerance persisted during the early hours of imbibition. Transgenic seeds also showed significantly improved resistance to controlled deterioration in a stable and transgene-dependent manner. Furthermore, overexpression of HaHSFA9 did not have detrimental effects on plant growth or development, including seed morphology and total seed yield. Our results agree with previous work tentatively associating HSP gene expression with phenotypes important for seed longevity. These findings might have implications for improving seed longevity in economically important crops. PMID:16998084

Effects of gravity on meiosis, fertilization and early embryogenesis in Caenorhabditis elegans

NASA Astrophysics Data System (ADS)

Sasagawa, Y.; Saito, Y.; Shimizu, M.; Ishioka, N.; Yamashita, M.; Takahashi, H.; Higashitani, A.

The embryonic development of the nematode Caenorhabditis elegans was examined under different gravitational conditions. The first cleavage plane in the 1-cell embryo was slid to some extent by re-orientation of liquid culture vessel, but the pattern and timing of cleavages were not affected. Under 100G of hypergravity condition with swing-centrifuge, the number of eggs laid from an adult hermaphrodite decreased and their hatching rate was drastically reduced. On the other hand, the embryonic development after fertilization normally occurred and grew to adulthood at more than 100G of hypergravity. When the adult hermaphrodites cultured under 100G of hypergravity transferred to a ground condition (1G), the newly fertilized embryos normally developed and their hatching rate was fully recovered. These results indicated that the reproductive process except spermatogenesis, oogenesis and embryogenesis after fertilization is impaired under 100G of hypergravity condition, and the effect is transient. Namely, the fertilization process including meiotic divisions I and II is sensitive to hypergravity in the nematode C. elegans.

The Modern Primitives: Applying New Technological Approaches to Explore the Biology of the Earliest Red Blood Cells

PubMed Central

Fraser, Stuart T.

2013-01-01

One of the most critical stages in mammalian embryogenesis is the independent production of the embryo's own circulating, functional red blood cells. Correspondingly, erythrocytes are the first cell type to become functionally mature during embryogenesis. Failure to achieve this invariably leads to in utero lethality. The recent application of technologies such as transcriptome analysis, flow cytometry, mutant embryo analysis, and transgenic fluorescent gene expression reporter systems has shed new light on the distinct erythroid lineages that arise early in development. Here, I will describe the similarities and differences between the distinct erythroid populations that must form for the embryo to survive. While much of the focus of this review will be the poorly understood primitive erythroid lineage, a discussion of other erythroid and hematopoietic lineages, as well as the cell types making up the different niches that give rise to these lineages, is essential for presenting an appropriate developmental context of these cells. PMID:24222861

Germ cell specification and ovary structure in the rotifer Brachionus plicatilis

PubMed Central

2010-01-01

Background The segregation of the germline from somatic tissues is an essential process in the development of all animals. Specification of the primordial germ cells (PGCs) takes place via different strategies across animal phyla; either specified early in embryogenesis by the inheritance of maternal determinants in the cytoplasm of the oocyte ('preformation') or selected later in embryonic development from undifferentiated precursors by a localized inductive signal ('epigenesis'). Here we investigate the specification and development of the germ cells in the rotifer Brachionus plicatilis, a member of the poorly-characterized superphyla Lophotrochozoa, by isolating the Brachionus homologues of the conserved germ cell markers vasa and nanos, and examining their expression using in situ hybridization. Results Bpvasa and Bpnos RNA expression have very similar distributions in the Brachionus ovary, showing ubiquitous expression in the vitellarium, with higher levels in the putative germ cell cluster. Bpvas RNA expression is present in freshly laid eggs, remaining ubiquitous in embryos until at least the 96 cell stage after which expression narrows to a small cluster of cells at the putative posterior of the embryo, consistent with the developing ovary. Bpnos RNA expression is also present in just-laid eggs but expression is much reduced by the four-cell stage and absent by the 16-cell stage. Shortly before hatching of the juvenile rotifer from the egg, Bpnos RNA expression is re-activated, located in a subset of posterior cells similar to those expressing Bpvas at the same stage. Conclusions The observed expression of vasa and nanos in the developing B. plicatilis embryo implies an epigenetic origin of primordial germ cells in Rotifer. PMID:20849649

Zebrafish Staufen1 and Staufen2 are required for the survival and migration of primordial germ cells.

PubMed

Ramasamy, Srinivas; Wang, Hui; Quach, Helen Ngoc Bao; Sampath, Karuna

2006-04-15

In sexually reproducing organisms, primordial germ cells (PGCs) give rise to the cells of the germ line, the gametes. In many animals, PGCs are set apart from somatic cells early during embryogenesis. Work in Drosophila, C. elegans, Xenopus, and zebrafish has shown that maternally provided localized cytoplasmic determinants specify the germ line in these organisms (Raz, E., 2003. Primordial germ-cell development: the zebrafish perspective. Nat. Rev., Genet. 4, 690--700; Santos, A.C., Lehmann, R., 2004. Germ cell specification and migration in Drosophila and beyond. Curr. Biol. 14, R578-R589). The Drosophila RNA-binding protein, Staufen is required for germ cell formation, and mutations in stau result in a maternal effect grandchild-less phenotype (Schupbach,T., Weischaus, E., 1989. Female sterile mutations on the second chromosome of Drosophila melanogaster:1. Maternal effect mutations. Genetics 121, 101-17). Here we describe the functions of two zebrafish Staufen-related proteins, Stau1 and Stau2. When Stau1 or Stau2 functions are compromised in embryos by injecting antisense morpholino modified oligonucleotides or dominant-negative Stau peptides, germ layer patterning is not affected. However, expression of the PGC marker vasa is not maintained. Furthermore, expression of a green fluorescent protein (GFP):nanos 3'UTR fusion protein in germ cells shows that PGC migration is aberrant, and the mis-migrating PGCs do not survive in Stau-compromised embryos. Stau2 is also required for survival of neurons in the central nervous system (CNS). These phenotypes are rescued by co-injection of Drosophila stau mRNA. Thus, staufen has an evolutionarily conserved function in germ cells. In addition, we have identified a function for Stau proteins in PGC migration.

Gap-Junctional communication between developing Drosophila muscles is essential for their normal development.

PubMed

Todman, M G; Baines, R A; Stebbings, L A; Davies, J A; Bacon, J P

1999-01-01

Recent experiments have demonstrated that a family of proteins, known as the innexins, are structural components of invertebrate gap junctions. The shaking-B (shak-B) locus of Drosophila encodes two members of this emerging family, Shak-B(lethal) and Shak-B(neural). This study focuses on the role of Shak-B gap junctions in the development of embryonic and larval muscle. During embryogenesis, shak-B transcripts are expressed in a subset of the somatic muscles; expression is strong in ventral oblique muscles (VO4-6) but only weak in ventral longitudinals (VL3 and 4). Carboxyfluorescein injected into VO4 of wild-type early stage 16 embryos spreads, via gap junctions, to label adjacent muscles, including VL3 and 4. In shak-B2 embryos (in which the shak-B(neural) function is disrupted), dye injected into VO4 fails to spread into other muscles. In the first instar larva, when dye coupling between muscles is no longer present, another effect of the shak-B2 mutation is revealed by whole-cell voltage clamp. In a calcium-free saline, only two voltage-activated potassium currents are present in wild-type muscles; a fast IA and a slow IK current. In shak-B2 larvae, these two currents are significantly reduced in magnitude in VO4 and 5, but remain normal in VL3. Expression of shak-B(neural) in a shak-B2 background fully rescues both dye coupling in embryonic muscle and whole-cell currents in first instar VO4 and 5. Our observations show that Shak-B(neural) is one of a set of embryonic gap-junction proteins, and that it is required for the normal temporal development of potassium currents in some larval muscles.

Rapid DNA Synthesis During Early Drosophila Embryogenesis Is Sensitive to Maternal Humpty Dumpty Protein Function.

PubMed

Lesly, Shera; Bandura, Jennifer L; Calvi, Brian R

2017-11-01

Problems with DNA replication cause cancer and developmental malformations. It is not fully understood how DNA replication is coordinated with development and perturbed in disease. We had previously identified the Drosophila gene humpty dumpty ( hd ), and showed that null alleles cause incomplete DNA replication, tissue undergrowth, and lethality. Animals homozygous for the missense allele, hd 272-9 , were viable, but adult females had impaired amplification of eggshell protein genes in the ovary, resulting in the maternal effects of thin eggshells and embryonic lethality. Here, we show that expression of an hd transgene in somatic cells of the ovary rescues amplification and eggshell synthesis but not embryo viability. The germline of these mothers remain mutant for the hd 272-9 allele, resulting in reduced maternal Hd protein and embryonic arrest during mitosis of the first few S/M nuclear cleavage cycles with chromosome instability and chromosome bridges. Epistasis analysis of hd with the rereplication mutation plutonium indicates that the chromosome bridges of hd embryos are the result of a failed attempt to segregate incompletely replicated sister chromatids. This study reveals that maternally encoded Humpty dumpty protein is essential for DNA replication and genome integrity during the little-understood embryonic S/M cycles. Moreover, the two hd 272-9 maternal-effect phenotypes suggest that ovarian gene amplification and embryonic cleavage are two time periods in development that are particularly sensitive to mild deficits in DNA replication function. This last observation has broader relevance for interpreting why mild mutations in the human ortholog of humpty dumpty and other DNA replication genes cause tissue-specific malformations of microcephalic dwarfisms. Copyright © 2017 by the Genetics Society of America.

Analysis of expression profiles of selected genes associated with the regenerative property and the receptivity to gene transfer during somatic embryogenesis in Triticum aestivum L.

PubMed

Delporte, Fabienne; Muhovski, Yordan; Pretova, Anna; Watillon, Bernard

2013-10-01

The physiological, biochemical and molecular mechanisms regulating the initiation of a regenerative pathway remain partially unknown. Efforts to identify the biological features that confer transformation ability, or the tendency of some cells to induce transgene silencing, would help to improve plant genetic engineering. The objective of our study was to monitor the evolution of plant cell competencies in relation to both in vitro tissue culture regeneration and the genetic transformation properties. We used a simple wheat regeneration procedure as an experimental model for studying the regenerative capacity of plant cells and their receptivity to direct gene transfer over the successive steps of the regenerative pathway. Target gene profiling studies and biochemical assays were conducted to follow some of the mechanisms triggered during the somatic-to-embryogenic transition (i.e. dedifferentiation, cell division activation, redifferentiation) and affecting the accessibility of plant cells to receive and stably express the exogenous DNA introduced by bombardment. Our results seem to indicate that the control of cell-cycle (S-phase) and host defense strategies can be crucial determinants of genetic transformation efficiency. The results from studies conducted at macro-, micro- and molecular scales are then integrated into a holistic approach that addresses the question of tissue culture and transgenesis competencies more broadly. Through this multilevel analysis we try to establish functional links between both regenerative capacity and transformation receptiveness, and thereby to provide a more global and integrated vision of both processes, at the core of defense/adaptive mechanisms and survival, between undifferentiated cell proliferation and organization.

Evolution and expression analysis reveal the potential role of the HD-Zip gene family in regulation of embryo abortion in grapes (Vitis vinifera L.).

PubMed

Li, Zhiqian; Zhang, Chen; Guo, Yurui; Niu, Weili; Wang, Yuejin; Xu, Yan

2017-09-21

The HD-Zip family has a diversity of functions during plant development. In this study, we identify 33 HD-Zip transcription factors in grape and detect their expressions in ovules and somatic embryos, as well as in various vegetative organs. A genome-wide survey for HD-Zip transcription factors in Vitis was conducted based on the 12 X grape genome (V. vinifera L.). A total of 33 members were identified and classified into four subfamilies (I-IV) based on phylogeny analysis with Arabidopsis, rice and maize. VvHDZs in the same subfamily have similar protein motifs and intron/exon structures. An evaluation of duplication events suggests several HD-Zip genes arose before the divergence of the grape and Arabidopsis lineages. The 33 members of HD-Zip were differentially expressed in ovules of the stenospermic grape, Thompson Seedless and of the seeded grape, Pinot noir. Most have higher expressions during ovule abortion in Thompson Seedless. In addition, transcripts of the HD-Zip family were also detected in somatic embryogenesis of Thompson Seedless and in different vegetative organs of Thompson Seedless at varying levels. Additionally, VvHDZ28 is located in the nucleus and had transcriptional activity consistent with the typical features of the HD-Zip family. Our results provide a foundation for future grape HD-Zip gene function research. The identification and expression profiles of the HD-Zip transcription factors in grape, reveal their diverse roles during ovule abortion and organ development. Our results lay a foundation for functional analysis of grape HDZ genes.

Cotyledonary somatic embryos of Pinus pinaster Ait. most closely resemble fresh, maturing cotyledonary zygotic embryos: biological, carbohydrate and proteomic analyses.

PubMed

Morel, Alexandre; Trontin, Jean-François; Corbineau, Françoise; Lomenech, Anne-Marie; Beaufour, Martine; Reymond, Isabelle; Le Metté, Claire; Ader, Kevin; Harvengt, Luc; Cadene, Martine; Label, Philippe; Teyssier, Caroline; Lelu-Walter, Marie-Anne

2014-11-01

Cotyledonary somatic embryos (SEs) of maritime pine are routinely matured for 12 weeks before being germinated and converted to plantlets. Although regeneration success is highly dependent on SEs quality, the date of harvesting is currently determined mainly on the basis of morphological features. This empirical method does not provide any accurate information about embryo quality with respect to storage compounds (proteins, carbohydrates). We first analyzed SEs matured for 10, 12 and 14 weeks by carrying out biological (dry weight, water content) and biochemical measurements (total protein and carbohydrate contents). No difference could be found between collection dates, suggesting that harvesting SEs after 12 weeks is appropriate. Cotyledonary SEs were then compared to various stages, from fresh to fully desiccated, in the development of cotyledonary zygotic embryos (ZEs). We identified profiles that were similar using hierarchical ascendant cluster analysis (HCA). Fresh and dehydrated ZEs could be distinguished, and SEs clustered with fresh ZEs. Both types of embryo exhibited similar carbohydrate and protein contents and signatures. This high level of similarity (94.5 %) was further supported by proteome profiling. Highly expressed proteins included storage, stress-related, late embryogenesis abundant and energy metabolism proteins. By comparing overexpressed proteins in developing and cotyledonary SEs or ZEs, some (23 proteins) could be identified as candidate biomarkers for the late, cotyledonary stage. This is the first report of useful generic protein markers for monitoring embryo development in maritime pine. Our results also suggest that improvements of SEs quality may be achieved if the current maturation conditions are refined.

Yolk formation in a stony coral Euphyllia ancora (Cnidaria, Anthozoa): insight into the evolution of vitellogenesis in nonbilaterian animals.

PubMed

Shikina, Shinya; Chen, Chieh-Jhen; Chung, Yi-Jou; Shao, Zi-Fan; Liou, Jhe-Yu; Tseng, Hua-Pin; Lee, Yan-Horn; Chang, Ching-Fong

2013-09-01

Vitellogenin (Vg) is a major yolk protein precursor in numerous oviparous animals. Numerous studies in bilateral oviparous animals have shown that Vg sequences are conserved across taxa and that Vgs are synthesized by somatic-cell lineages, transported to and accumulated in oocytes, and eventually used for supporting embryogenesis. In nonbilateral animals (Polifera, Cnidaria, and Ctenophora), which are regarded as evolutionarily primitive, although Vg cDNA has been identified in 2 coral species from Cnidaria, relatively little is known about the characteristics of yolk formation in their bodies. To address this issue, we identified and characterized 2 cDNA encoding yolk proteins, Vg and egg protein (Ep), in the stony coral Euphyllia ancora. RT-PCR analysis revealed that expression levels of both Vg and Ep increased in the female colonies as coral approached the spawning season. In addition, high levels of both Vg and Ep transcripts were detected in the putative ovarian tissue, as determined by tissue distribution analysis. Further analyses using mRNA in situ hybridization and immunohistochemistry determined that, within the putative ovarian tissue, these yolk proteins are synthesized in the mesenterial somatic cells but not in oocytes themselves. Furthermore, Vg proteins that accumulated in eggs were most likely consumed during the coral embryonic development, as assessed by immunoblotting. The characteristics of Vg that we identified in corals were somewhat similar to those of Vg in bilaterian oviparous animals, raising the hypothesis that such characteristics were likely present in the oogenesis of some common ancestor prior to divergence of the cnidarian and bilaterian lineages.

Somatic Host Cell Alterations in HPV Carcinogenesis

PubMed Central

Litwin, Tamara R.; Clarke, Megan A.; Dean, Michael; Wentzensen, Nicolas

2017-01-01

High-risk human papilloma virus (HPV) infections cause cancers in different organ sites, most commonly cervical and head and neck cancers. While carcinogenesis is initiated by two viral oncoproteins, E6 and E7, increasing evidence shows the importance of specific somatic events in host cells for malignant transformation. HPV-driven cancers share characteristic somatic changes, including apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC)-driven mutations and genomic instability leading to copy number variations and large chromosomal rearrangements. HPV-associated cancers have recurrent somatic mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and phosphatase and tensin homolog (PTEN), human leukocyte antigen A and B (HLA-A and HLA-B)-A/B, and the transforming growth factor beta (TGFβ) pathway, and rarely have mutations in the tumor protein p53 (TP53) and RB transcriptional corepressor 1 (RB1) tumor suppressor genes. There are some variations by tumor site, such as NOTCH1 mutations which are primarily found in head and neck cancers. Understanding the somatic events following HPV infection and persistence can aid the development of early detection biomarkers, particularly when mutations in precancers are characterized. Somatic mutations may also influence prognosis and treatment decisions. PMID:28771191

Somatic Host Cell Alterations in HPV Carcinogenesis.

PubMed

Litwin, Tamara R; Clarke, Megan A; Dean, Michael; Wentzensen, Nicolas

2017-08-03

High-risk human papilloma virus (HPV) infections cause cancers in different organ sites, most commonly cervical and head and neck cancers. While carcinogenesis is initiated by two viral oncoproteins, E6 and E7, increasing evidence shows the importance of specific somatic events in host cells for malignant transformation. HPV-driven cancers share characteristic somatic changes, including apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC)-driven mutations and genomic instability leading to copy number variations and large chromosomal rearrangements. HPV-associated cancers have recurrent somatic mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha ( PIK3CA ) and phosphatase and tensin homolog ( PTEN ), human leukocyte antigen A and B ( HLA-A and HLA-B ) -A/B , and the transforming growth factor beta (TGFβ) pathway, and rarely have mutations in the tumor protein p53 ( TP53 ) and RB transcriptional corepressor 1 ( RB1 ) tumor suppressor genes. There are some variations by tumor site, such as NOTCH1 mutations which are primarily found in head and neck cancers. Understanding the somatic events following HPV infection and persistence can aid the development of early detection biomarkers, particularly when mutations in precancers are characterized. Somatic mutations may also influence prognosis and treatment decisions.

Intestinal development and differentiation

PubMed Central

Noah, Taeko K.; Donahue, Bridgitte; Shroyer, Noah F.

2011-01-01

In this review, we present an overview of intestinal development and cellular differentiation of the intestinal epithelium. The review is separated into two sections: Section one summarizes organogenesis of the small and large intestines, including endoderm and gut tube formation in early embryogenesis, villus morphogenesis, and crypt formation. Section two reviews cell fate specification and differentiation of each cell type within the intestinal epithelium. Growth factor and transcriptional networks that regulate these developmental processes are summarized. PMID:21978911

Bloom syndrome: a mendelian prototype of somatic mutational disease.

PubMed

German, J

1993-11-01

Spontaneous mutations in human somatic cells occur far more often than normal in individuals with Bloom syndrome. The basis for understanding these mutations and their developmental consequences emerges from examination of BS at the molecular, cellular, and clinical levels. The major clinical feature of BS, proportional dwarfism, as well as its major clinical complication, an exceptionally early emergence of neoplasia of the types and sites that affect the general population, are attributable to the excessive occurrence of mutations in somatic cells. Here, the following aspects of BS are discussed: (i) the BS phenotype; (ii) neoplasia in BS, including the means--the Bloom's Syndrome Registry--by which the significant risk for diverse sites and types of cancer in these patients was revealed; (iii) the biological basis for the cancer proneness of BS; and, finally, (iv) the significance for both basic human biology and clinical medicine of BS as the prototype of somatic mutational disease.

Development of the Neurochemical Architecture of the Central Complex

PubMed Central

Boyan, George S.; Liu, Yu

2016-01-01

The central complex represents one of the most conspicuous neuroarchitectures to be found in the insect brain and regulates a wide repertoire of behaviors including locomotion, stridulation, spatial orientation and spatial memory. In this review article, we show that in the grasshopper, a model insect system, the intricate wiring of the fan-shaped body (FB) begins early in embryogenesis when axons from the first progeny of four protocerebral stem cells (called W, X, Y, Z, respectively) in each brain hemisphere establish a set of tracts to the primary commissural system. Decussation of subsets of commissural neurons at stereotypic locations across the brain midline then establishes a columnar neuroarchitecture in the FB which is completed during embryogenesis. Examination of the expression patterns of various neurochemicals in the central complex including neuropeptides, a neurotransmitter and the gas nitric oxide (NO), show that these appear progressively and in a substance-specific manner during embryogenesis. Each neuroactive substance is expressed by neurons located at stereotypic locations in a given central complex lineage, confirming that the stem cells are biochemically multipotent. The organization of axons expressing the various neurochemicals within the central complex is topologically related to the location, and hence birthdate, of the neurons within the lineages. The neurochemical expression patterns within the FB are layered, and so reflect the temporal topology present in the lineages. This principle relates the neuroanatomical to the neurochemical architecture of the central complex and so may provide insights into the development of adaptive behaviors. PMID:27630548

Genetic analysis of fat-to-protein ratio, milk yield and somatic cell score of Holstein cows in Japan in the first three lactations by using a random regression model.

PubMed

Nishiura, Akiko; Sasaki, Osamu; Aihara, Mitsuo; Takeda, Hisato; Satoh, Masahiro

2015-12-01

We estimated the genetic parameters of fat-to-protein ratio (FPR) and the genetic correlations between FPR and milk yield or somatic cell score in the first three lactations in dairy cows. Data included 3,079,517 test-day records of 201,138 Holstein cows in Japan from 2006 to 2011. Genetic parameters were estimated with a multiple-trait random regression model in which the records within and between parities were treated as separate traits. The phenotypic values of FPR increased soon after parturition and peaked at 10 to 20 days in milk, then decreased slowly in mid- and late lactation. Heritability estimates for FPR yielded moderate values. Genetic correlations of FPR among parities were low in early lactation. Genetic correlations between FPR and milk yield were positive and low in early lactation, but only in the first lactation. Genetic correlations between FPR and somatic cell score were positive in early lactation and decreased to become negative in mid- to late lactation. By using these results for genetic evaluation it should be possible to improve energy balance in dairy cows. © 2015 Japanese Society of Animal Science.

Transcriptome analyses of rhesus monkey preimplantation embryos reveal a reduced capacity for DNA double-strand break repair in primate oocytes and early embryos

PubMed Central

Wang, Xinyi; Liu, Denghui; He, Dajian; Suo, Shengbao; Xia, Xian; He, Xiechao; Han, Jing-Dong J.; Zheng, Ping

2017-01-01

Preimplantation embryogenesis encompasses several critical events including genome reprogramming, zygotic genome activation (ZGA), and cell-fate commitment. The molecular basis of these processes remains obscure in primates in which there is a high rate of embryo wastage. Thus, understanding the factors involved in genome reprogramming and ZGA might help reproductive success during this susceptible period of early development and generate induced pluripotent stem cells with greater efficiency. Moreover, explaining the molecular basis responsible for embryo wastage in primates will greatly expand our knowledge of species evolution. By using RNA-seq in single and pooled oocytes and embryos, we defined the transcriptome throughout preimplantation development in rhesus monkey. In comparison to archival human and mouse data, we found that the transcriptome dynamics of monkey oocytes and embryos were very similar to those of human but very different from those of mouse. We identified several classes of maternal and zygotic genes, whose expression peaks were highly correlated with the time frames of genome reprogramming, ZGA, and cell-fate commitment, respectively. Importantly, comparison of the ZGA-related network modules among the three species revealed less robust surveillance of genomic instability in primate oocytes and embryos than in rodents, particularly in the pathways of DNA damage signaling and homology-directed DNA double-strand break repair. This study highlights the utility of monkey models to better understand the molecular basis for genome reprogramming, ZGA, and genomic stability surveillance in human early embryogenesis and may provide insights for improved homologous recombination-mediated gene editing in monkey. PMID:28223401

Cancer heterogeneity: converting a limitation into a source of biologic information.

PubMed

Rübben, Albert; Araujo, Arturo

2017-09-08

Analysis of spatial and temporal genetic heterogeneity in human cancers has revealed that somatic cancer evolution in most cancers is not a simple linear process composed of a few sequential steps of mutation acquisitions and clonal expansions. Parallel evolution has been observed in many early human cancers resulting in genetic heterogeneity as well as multilineage progression. Moreover, aneuploidy as well as structural chromosomal aberrations seems to be acquired in a non-linear, punctuated mode where most aberrations occur at early stages of somatic cancer evolution. At later stages, the cancer genomes seem to get stabilized and acquire only few additional rearrangements. While parallel evolution suggests positive selection of driver mutations at early stages of somatic cancer evolution, stabilization of structural aberrations at later stages suggests that negative selection takes effect when cancer cells progressively lose their tolerance towards additional mutation acquisition. Mixing of genetically heterogeneous subclones in cancer samples reduces sensitivity of mutation detection. Moreover, driver mutations present only in a fraction of cancer cells are more likely to be mistaken for passenger mutations. Therefore, genetic heterogeneity may be considered a limitation negatively affecting detection sensitivity of driver mutations. On the other hand, identification of subclones and subclone lineages in human cancers may lead to a more profound understanding of the selective forces which shape somatic cancer evolution in human cancers. Identification of parallel evolution by analyzing spatial heterogeneity may hint to driver mutations which might represent additional therapeutic targets besides driver mutations present in a monoclonal state. Likewise, stabilization of cancer genomes which can be identified by analyzing temporal genetic heterogeneity might hint to genes and pathways which have become essential for survival of cancer cell lineages at later stages of cancer evolution. These genes and pathways might also constitute patient specific therapeutic targets.

Adverse Childhood Experiences and Child Health in Early Adolescence

PubMed Central

Flaherty, Emalee G.; Thompson, Richard; Dubowitz, Howard; Harvey, Elizabeth M; English, Diana J.; Everson, Mark D.; Proctor, Laura J.; Runyan, Desmond K.

2013-01-01

Objective 1) Examine the relationship between previous adverse childhood experiences and somatic complaints and health problems in early adolescence, and 2) examine the role of the timing of adverse exposures. Design Prospective analysis of the Longitudinal Studies of Child Abuse and Neglect interview data when children were 4, 6, 8, 12 and 14 years old. Setting Children reported or at risk for maltreatment in the South, East, Midwest, Northwest, and Southwest United States LONGSCAN sites Participants 933 children. Main Exposures Eight categories of adversity (psychological maltreatment, physical abuse, sexual abuse, neglect, caregiver’s substance use/alcohol abuse, caregiver’s depressive symptoms, caregiver treated violently, and criminal behavior by household member) experienced during the first 6 years of life, the second six years of life, the most recent 2 years, and overall adversity Outcome Measures Child health problems including poor health, illness requiring a doctor, somatic complaints and any health problem at age 14. Results More than 90% of the youth had experienced an adverse childhood event by age 14. There was a graded relationship between adverse childhood exposures and any health problem, while 2 and ≥3 adverse exposures were associated with somatic complaints. Recent adversity uniquely predicted poor health, somatic complaints and any health problem. Conclusions Childhood adversities, particularly recent adversities, already impair the health of young adolescents. Increased efforts to prevent and mitigate these experiences may improve the health of adolescents and adults. PMID:23645114

A modified culture method significantly improves the development of mouse somatic cell nuclear transfer embryos.

PubMed

Dai, Xiangpeng; Hao, Jie; Zhou, Qi

2009-08-01

Many strategies have been established to improve the efficiency of somatic cell nuclear transfer (SCNT), but relatively few focused on improving culture conditions. The effect of different culture media on preimplantation development of mouse nuclear transfer embryos was investigated. A modified sequential media method, named D media (M16/KSOM and CZB-EG/KSOM), was successfully established that significantly improves SCNT embryo development. Our result demonstrated that while lacking any adverse effect on in vivo fertilized embryos, the D media dramatically improves the blastocyst development of SCNT embryos compared with other commonly used media, including KSOM, M16, CZB, and alphaMEM. Specifically, the rate of blastocyst formation was 62.3% for D1 (M16/KSOM) versus 10-30% for the other media. An analysis of media components indicated that removing EDTA and glutamine from the media can be beneficial for early SCNT embryo development. Our results suggest that in vitro culture environment plays an important role in somatic cell reprogramming, and D media represent the most efficient culture method reported to date to support mouse SCNT early embryo development in vitro.

The Treacher Collins syndrome (TCOF1) gene product is involved in ribosomal DNA gene transcription by interacting with upstream binding factor.

PubMed

Valdez, Benigno C; Henning, Dale; So, Rolando B; Dixon, Jill; Dixon, Michael J

2004-07-20

Treacher Collins syndrome (TCS) is an autosomal dominant disorder characterized by an abnormality of craniofacial development that arises during early embryogenesis. TCS is caused by mutations in the gene TCOF1, which encodes the nucleolar phosphoprotein treacle. Even though the genetic alterations causing TCS have been uncovered, the mechanism underlying its pathogenesis and the function of treacle remain unknown. Here, we show that treacle is involved in ribosomal DNA gene transcription by interacting with upstream binding factor (UBF). Immunofluorescence labeling shows treacle and UBF colocalize to specific nucleolar organizer regions and cosegregate within nucleolar caps of actinomycin d-treated HeLa cells. Biochemical analysis shows the association of treacle and UBF with chromatin. Immunoprecipitation and the yeast two-hybrid system both suggest physical interaction of the two nucleolar phosphoproteins. Down-regulation of treacle expression using specific short interfering RNA results in inhibition of ribosomal DNA transcription and cell growth. A similar correlation is observed in Tcof(+/-) mouse embryos that exhibit craniofacial defects and growth retardation. Thus, treacle haploinsufficiency in TCS patients might result in abnormal development caused by inadequate ribosomal RNA production in the prefusion neural folds during the early stages of embryogenesis. The elucidation of a physiological function of treacle provides important information of relevance to the molecular dissection of the biochemical pathology of TCS.

The Treacher Collins syndrome (TCOF1) gene product is involved in ribosomal DNA gene transcription by interacting with upstream binding factor

PubMed Central

Valdez, Benigno C.; Henning, Dale; So, Rolando B.; Dixon, Jill; Dixon, Michael J.

2004-01-01

Treacher Collins syndrome (TCS) is an autosomal dominant disorder characterized by an abnormality of craniofacial development that arises during early embryogenesis. TCS is caused by mutations in the gene TCOF1, which encodes the nucleolar phosphoprotein treacle. Even though the genetic alterations causing TCS have been uncovered, the mechanism underlying its pathogenesis and the function of treacle remain unknown. Here, we show that treacle is involved in ribosomal DNA gene transcription by interacting with upstream binding factor (UBF). Immunofluorescence labeling shows treacle and UBF colocalize to specific nucleolar organizer regions and cosegregate within nucleolar caps of actinomycin d-treated HeLa cells. Biochemical analysis shows the association of treacle and UBF with chromatin. Immunoprecipitation and the yeast two-hybrid system both suggest physical interaction of the two nucleolar phosphoproteins. Down-regulation of treacle expression using specific short interfering RNA results in inhibition of ribosomal DNA transcription and cell growth. A similar correlation is observed in Tcof+/- mouse embryos that exhibit craniofacial defects and growth retardation. Thus, treacle haploinsufficiency in TCS patients might result in abnormal development caused by inadequate ribosomal RNA production in the prefusion neural folds during the early stages of embryogenesis. The elucidation of a physiological function of treacle provides important information of relevance to the molecular dissection of the biochemical pathology of TCS. PMID:15249688

The Forms and Sources of Cytokinins in Developing White Lupine Seeds and Fruits1

PubMed Central

Emery, R.J. Neil; Ma, Qifu; Atkins, Craig A.

2000-01-01

A comprehensive range of cytokinins (CK) was identified and quantified by gas chromatography-mass spectrometry in tissues of and in xylem and phloem serving developing white lupine (Lupinus albus) fruits. Analyses were initiated at anthesis and included stages of podset, embryogenesis, and seed filling up to physiological maturation 77 d post anthesis (DPA). In the first 10 DPA, fertilized ovaries destined to set pods accumulated CK. The proportion of cis-CK:trans-CK isomers was initially 10:1 but declined to less than 1:1. In ovaries destined to abort, the ratio of cis-isomers to trans-isomers remained high. During early podset, accumulation of CK (30–40 pmol ovary−1) was accounted for by xylem and phloem translocation, both containing more than 90% cis-isomers. During embryogenesis and early seed filling (40–46 DPA), translocation accounted for 1% to 14% of the increases of CK in endosperm (20 nmol fruit−1) and seed coat (15 nmol fruit−1), indicating synthesis in situ. High CK concentrations in seeds (0.6 μmol g−1 fresh weight) were transient, declining rapidly to less than 1% of maximum levels by physiological maturity. These data pose new questions about the localization and timing of CK synthesis, the significance of translocation, and the role(s) of CK forms in reproductive development. PMID:10938375

Maternal Setdb1 Is Required for Meiotic Progression and Preimplantation Development in Mouse.

PubMed

Kim, Jeesun; Zhao, Hongbo; Dan, Jiameng; Kim, Soojin; Hardikar, Swanand; Hollowell, Debra; Lin, Kevin; Lu, Yue; Takata, Yoko; Shen, Jianjun; Chen, Taiping

2016-04-01

Oocyte meiotic progression and maternal-to-zygote transition are accompanied by dynamic epigenetic changes. The functional significance of these changes and the key epigenetic regulators involved are largely unknown. Here we show that Setdb1, a lysine methyltransferase, controls the global level of histone H3 lysine 9 di-methyl (H3K9me2) mark in growing oocytes. Conditional deletion of Setdb1 in developing oocytes leads to meiotic arrest at the germinal vesicle and meiosis I stages, resulting in substantially fewer mature eggs. Embryos derived from these eggs exhibit severe defects in cell cycle progression, progressive delays in preimplantation development, and degeneration before reaching the blastocyst stage. Rescue experiments by expressing wild-type or inactive Setdb1 in Setdb1-deficient oocytes suggest that the catalytic activity of Setdb1 is essential for meiotic progression and early embryogenesis. Mechanistically, up-regulation of Cdc14b, a dual-specificity phosphatase that inhibits meiotic progression, greatly contributes to the meiotic arrest phenotype. Setdb1 deficiency also leads to derepression of transposons and increased DNA damage in oocytes, which likely also contribute to meiotic defects. Thus, Setdb1 is a maternal-effect gene that controls meiotic progression and is essential for early embryogenesis. Our results uncover an important link between the epigenetic machinery and the major signaling pathway governing meiotic progression.

Mutations in the DNA methyltransferase gene DNMT3A cause an overgrowth syndrome with intellectual disability.

PubMed

Tatton-Brown, Katrina; Seal, Sheila; Ruark, Elise; Harmer, Jenny; Ramsay, Emma; Del Vecchio Duarte, Silvana; Zachariou, Anna; Hanks, Sandra; O'Brien, Eleanor; Aksglaede, Lise; Baralle, Diana; Dabir, Tabib; Gener, Blanca; Goudie, David; Homfray, Tessa; Kumar, Ajith; Pilz, Daniela T; Selicorni, Angelo; Temple, I Karen; Van Maldergem, Lionel; Yachelevich, Naomi; van Montfort, Robert; Rahman, Nazneen

2014-04-01

Overgrowth disorders are a heterogeneous group of conditions characterized by increased growth parameters and other variable clinical features such as intellectual disability and facial dysmorphism. To identify new causes of human overgrowth, we performed exome sequencing in ten proband-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations by sequencing DNMT3A in a further 142 individuals with overgrowth. The mutations alter residues in functional DNMT3A domains, and protein modeling suggests that they interfere with domain-domain interactions and histone binding. Similar mutations were not present in 1,000 UK population controls (13/152 cases versus 0/1,000 controls; P < 0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and greater height. DNMT3A encodes a DNA methyltransferase essential for establishing methylation during embryogenesis and is commonly somatically mutated in acute myeloid leukemia. Thus, DNMT3A joins an emerging group of epigenetic DNA- and histone-modifying genes associated with both developmental growth disorders and hematological malignancies.

Loss-of-function mutations in LEMD3 result in osteopoikilosis, Buschke-Ollendorff syndrome and melorheostosis.

PubMed

Hellemans, Jan; Preobrazhenska, Olena; Willaert, Andy; Debeer, Philippe; Verdonk, Peter C M; Costa, Teresa; Janssens, Katrien; Menten, Bjorn; Van Roy, Nadine; Vermeulen, Stefan J T; Savarirayan, Ravi; Van Hul, Wim; Vanhoenacker, Filip; Huylebroeck, Danny; De Paepe, Anne; Naeyaert, Jean-Marie; Vandesompele, Jo; Speleman, Frank; Verschueren, Kristin; Coucke, Paul J; Mortier, Geert R

2004-11-01

Osteopoikilosis, Buschke-Ollendorff syndrome (BOS) and melorheostosis are disorders characterized by increased bone density. The occurrence of one or more of these phenotypes in the same individual or family suggests that these entities might be allelic. We collected data from three families in which affected individuals had osteopoikilosis with or without manifestations of BOS or melorheostosis. A genome-wide linkage analysis in these families, followed by the identification of a microdeletion in an unrelated individual with these diseases, allowed us to map the gene that is mutated in osteopoikilosis. All the affected individuals that we investigated were heterozygous with respect to a loss-of-function mutation in LEMD3 (also called MAN1), which encodes an inner nuclear membrane protein. A somatic mutation in the second allele of LEMD3 could not be identified in fibroblasts from affected skin of an individual with BOS and an individual with melorheostosis. XMAN1, the Xenopus laevis ortholog, antagonizes BMP signaling during embryogenesis. In this study, LEMD3 interacted with BMP and activin-TGFbeta receptor-activated Smads and antagonized both signaling pathways in human cells.

Mutations in the DNA methyltransferase gene, DNMT3A, cause an overgrowth syndrome with intellectual disability

PubMed Central

Tatton-Brown, Katrina; Seal, Sheila; Ruark, Elise; Harmer, Jenny; Ramsay, Emma; del Vecchio Duarte, Silvana; Zachariou, Anna; Hanks, Sandra; O’Brien, Eleanor; Aksglaede, Lise; Baralle, Diana; Dabir, Tabib; Gener, Blanca; Goudie, David; Homfray, Tessa; Kumar, Ajith; Pilz, Daniela T; Selicorni, Angelo; Temple, I Karen; Van Maldergem, Lionel; Yachelevich, Naomi; van Montfort, Robert; Rahman, Nazneen

2014-01-01

Overgrowth disorders are a heterogeneous group of conditions characterised by increased growth parameters and variable other clinical features, such as intellectual disability and facial dysmorphism1. To identify novel causes of human overgrowth we performed exome sequencing in 10 proband-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations through DNMT3A sequencing of a further 142 individuals with overgrowth. The mutations were all located in functional DNMT3A domains and protein modelling suggests they interfere with domain-domain interactions and histone binding. No similar mutations were present in 1000 UK population controls (13/152 vs 0/1000; P<0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and increased height. DNMT3A encodes a key methyltransferase essential for establishing the methylation imprint in embryogenesis and is commonly somatically mutated in acute myeloid leukaemia2-4. Thus DNMT3A joins an emerging group of epigenetic DNA and histone modifying genes associated with both developmental growth disorders and haematological malignancies5. PMID:24614070

Recurrent DNA inversion rearrangements in the human genome

PubMed Central

Flores, Margarita; Morales, Lucía; Gonzaga-Jauregui, Claudia; Domínguez-Vidaña, Rocío; Zepeda, Cinthya; Yañez, Omar; Gutiérrez, María; Lemus, Tzitziki; Valle, David; Avila, Ma. Carmen; Blanco, Daniel; Medina-Ruiz, Sofía; Meza, Karla; Ayala, Erandi; García, Delfino; Bustos, Patricia; González, Víctor; Girard, Lourdes; Tusie-Luna, Teresa; Dávila, Guillermo; Palacios, Rafael

2007-01-01

Several lines of evidence suggest that reiterated sequences in the human genome are targets for nonallelic homologous recombination (NAHR), which facilitates genomic rearrangements. We have used a PCR-based approach to identify breakpoint regions of rearranged structures in the human genome. In particular, we have identified intrachromosomal identical repeats that are located in reverse orientation, which may lead to chromosomal inversions. A bioinformatic workflow pathway to select appropriate regions for analysis was developed. Three such regions overlapping with known human genes, located on chromosomes 3, 15, and 19, were analyzed. The relative proportion of wild-type to rearranged structures was determined in DNA samples from blood obtained from different, unrelated individuals. The results obtained indicate that recurrent genomic rearrangements occur at relatively high frequency in somatic cells. Interestingly, the rearrangements studied were significantly more abundant in adults than in newborn individuals, suggesting that such DNA rearrangements might start to appear during embryogenesis or fetal life and continue to accumulate after birth. The relevance of our results in regard to human genomic variation is discussed. PMID:17389356

In vitro propagation of ginger (Zingiber officinale Rosc.) through direct organogenesis: a review.

PubMed

Seran, Thayamini H

2013-12-15

Ginger (Zingiber officinale Rosc.) is a perennial herb. It belongs to the family Zingiberaceae and commercially cultivated in most tropical regions of the world. The underground rhizomes are the planting materials in a conventional propagation of ginger however it has a low multiplication rate. It is known that there are possible methods are available for rapid vegetative propagation of ginger through direct organogenesis or somatic embryogenesis under in vitro conditions but it is necessary to find the best protocol for in vitro multiplication of ginger. Limited studies on the tissue culture technology of ginger are available in Sri Lanka. However, significant efforts have been made in the procedure for in vitro micropropagation in the other ginger growing countries. The available literature with respect to in vitro plant regeneration has been perused and this review mainly focused on the in vitro propagation via direct organogenesis from rhizome buds or shoot tips of ginger often used as explants. This review article may be an appropriate and effective guidance for establishing in vitro cultures and subsequent production of in vitro plantlets in clonal propagation of ginger.

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.

Selective chromatid segregation mechanism for Bruchus wings piebald color.

PubMed

Klar, Amar J S

2015-01-01

The mechanisms of asymmetric organ development have been under intensive investigation for years, yet the proposed mechanisms remain controversial (1-3). The female Bruchus quadrimaculatus beetle insect develops two black-colored spots bilaterally located on each upper elytra wing by an unknown mechanism. Fifty percent of the P (for piebald, two colors) gene homozygous mutant insects, described in 1925, had a normal left elytrum (with two black spots) and an abnormal right elytrum (with two red spots) and the balance supported the converse lateralized pigment arrangement (4). Rather than supporting the conventional morphogen model for the wings pigmentation development, their biological origin is explained here with the somatic strand-specific epigenetic imprinting and selective sister chromatid segregation (SSIS) mechanism (5). We propose that the P gene product performs the selective sister chromatid segregation function to produce symmetric cell division of a specific cell during embryogenesis to result in the bilateral symmetric development of elytra black color spots and that the altered chromatid segregation pattern of the mutant causes asymmetric cell division to confer the piebald phenotype. 

Allosteric receptor activation by the plant peptide hormone phytosulfokine.

PubMed

Wang, Jizong; Li, Hongju; Han, Zhifu; Zhang, Heqiao; Wang, Tong; Lin, Guangzhong; Chang, Junbiao; Yang, Weicai; Chai, Jijie

2015-09-10

Phytosulfokine (PSK) is a disulfated pentapeptide that has a ubiquitous role in plant growth and development. PSK is perceived by its receptor PSKR, a leucine-rich repeat receptor kinase (LRR-RK). The mechanisms underlying the recognition of PSK, the activation of PSKR and the identity of the components downstream of the initial binding remain elusive. Here we report the crystal structures of the extracellular LRR domain of PSKR in free, PSK- and co-receptor-bound forms. The structures reveal that PSK interacts mainly with a β-strand from the island domain of PSKR, forming an anti-β-sheet. The two sulfate moieties of PSK interact directly with PSKR, sensitizing PSKR recognition of PSK. Supported by biochemical, structural and genetic evidence, PSK binding enhances PSKR heterodimerization with the somatic embryogenesis receptor-like kinases (SERKs). However, PSK is not directly involved in PSKR-SERK interaction but stabilizes PSKR island domain for recruitment of a SERK. Our data reveal the structural basis for PSKR recognition of PSK and allosteric activation of PSKR by PSK, opening up new avenues for the design of PSKR-specific small molecules.

Establishment of stem cell lines from nuclear transferred and parthenogenetically activated mouse oocytes for therapeutic cloning.

PubMed

Ju, Jin Young; Park, Chun Young; Gupta, Mukesh Kumar; Uhm, Sang Jun; Paik, Eun Chan; Ryoo, Zae Young; Cho, Youl Hee; Chung, Kil Saeng; Lee, Hoon Taek

2008-05-01

To establish embryonic stem cell lines from nuclear transfer of somatic cell nuclei isolated from the same oocyte donor and from parthenogenetic activation. The study also evaluated the effect of the micromanipulation procedure on the outcome of somatic cell nuclear transfer in mice. Randomized, prospective study. Hospital-based assisted reproductive technology laboratory. F(1) (C57BL/6 x 129P3/J) mice. Metaphase II-stage oocytes were either parthenogenetically activated or nuclear transferred with cumulus cell nuclei or parthenogenetically activated after a sham-manipulation procedure. Embryogenesis and embryonic stem cell establishment. The development rate to morula/blastocyst of nuclear transferred oocytes (27.9% +/- 5.9%) was significantly lower than that of the sham-manipulated (84.1% +/- 5.6%) or parthenogenetic (98.6% +/- 1.4%) groups. A sharp decrease in cleavage potential was obvious in the two- to four-cell transition for the nuclear transferred embryos (79.0% +/- 4.6% and 43.3% +/- 5.0%), implying incomplete nuclear reprogramming in arrested oocytes. However, the cleavage, as well as the development rate, of parthenogenetic and sham-manipulated groups did not differ significantly. The embryonic stem cell line establishment rate was higher from parthenogenetically activated oocytes (15.7%) than nuclear transferred (4.3%) or sham-manipulated oocytes (12.5%). Cell colonies from all groups displayed typical morphology of mice embryonic stem cells and could be maintained successfully with undifferentiated morphology after continuous proliferation for more than 120 passages still maintaining normal karyotype. All these cells were positive for mice embryonic stem cell markers such as Oct-4 and SSEA-1 based on immunocytochemistry and reverse transcriptase-polymerase chain reaction. The clonal origin of the ntES cell line and the parthenogenetic embryonic stem cell lines were confirmed by polymerase chain reaction analysis of the polymorphic markers. Blastocyst injection experiments demonstrated that these lines contributed to resulting chimeras and are germ-line competent. We report the establishment of ntES cell lines from somatic cells isolated from same individual. Our data also suggest that embryo micromanipulation procedure during the nuclear transfer procedure influences the developmental ability and embryonic stem cell establishment rate of nuclear transferred embryos.

Novel functions for the RNA-binding protein ETR-1 in Caenorhabditis elegans reproduction and engulfment of germline apoptotic cell corpses

PubMed Central

Boateng, Ruby; Nguyen, Ken C.Q.; Hall, David H.; Golden, Andy; Allen, Anna K.

2017-01-01

RNA-binding proteins (RBPs) are essential regulators of gene expression that act through a variety of mechanisms to ensure the proper post-transcriptional regulation of their target RNAs. RBPs in multiple species have been identified as playing crucial roles during development and as having important functions in various adult organ systems, including the heart, nervous, muscle, and reproductive systems. ETR-1, a highly conserved ELAV-Type RNA-binding protein belonging to the CELF/Bruno protein family, has been previously reported to be involved in C. elegans muscle development. Animals depleted of ETR-1 have been previously characterized as arresting at the two-fold stage of embryogenesis. In this study, we show that ETR-1 is expressed in the hermaphrodite somatic gonad and germ line, and that reduction of ETR-1 via RNA interference (RNAi) results in reduced hermaphrodite fecundity. Detailed characterization of this fertility defect indicates that ETR-1 is required in both the somatic tissue and the germ line to ensure wild-type reproductive levels. Additionally, the ability of ETR-1 depletion to suppress the published WEE-1.3-depletion infertility phenotype is dependent on ETR-1 being reduced in the soma. Within the germline of etr-1(RNAi) hermaphrodite animals, we observe a decrease in average oocyte size and an increase in the number of germline apoptotic cell corpses as evident by an increased number of CED-1::GFP and acridine orange positive apoptotic germ cells. Transmission Electron Microscopy (TEM) studies confirm the significant increase in apoptotic cells in ETR-1-depleted animals, and reveal a failure of the somatic gonadal sheath cells to properly engulf dying germ cells in etr-1(RNAi) animals. Through investigation of an established engulfment pathway in C. elegans, we demonstrate that co-depletion of CED-1 and ETR-1 suppresses both the reduced fecundity and the increase in the number of apoptotic cell corpses observed in etr-1(RNAi) animals. Combined, this data identifies a novel role for ETR-1 in hermaphrodite gametogenesis and in the process of engulfment of germline apoptotic cell corpses. PMID:28648844

Patterns of protein synthesis in oocytes and early embryos of Rana esculenta complex.

PubMed

Chen, P S; Stumm-Zollinger, E

1986-01-01

We have used isotopic labelling and both one-and two-dimensional electrophoretic procedures to analyse the protien synthesis patterns in oocytes and early embryos of three phenotypes of the European green frogs. The results demonstrated that protein patterns of Rana ridibunda and R. esculenta are identical, but that they differ from those of R. lessonae. Progeny of the lethal cross R. esculenta × R. esculenta showed a distinct delay in the appearance of stage-specific proteins during early embryogenesis. The heat-shock response of R. ridibunda and R. esculenta oocytes was found to be identical, but different from that of Xenopus laevis. The implications of these findings, with respect to hybridogenesis in R. esculenta complex and variations in the regulations of heat shock genes in different amphibian species, are discussed.

Nutrient-gene interactions in early pregnancy: a vascular hypothesis.

PubMed

Steegers-Theunissen, R P M; Steegers, E A P

2003-02-10

It is hypothesized that the following periconceptional and early pregnancy nutrient-gene interactions link vascular-related reproductive complications and cardiovascular diseases in adulthood: (1) Maternal and paternal genetically controlled nutrient status affects the quality of gametes and fertilization capacity; (2) The embryonic genetic constitution, derived from both parents, and the maternal genetically controlled nutrient environment determine embryogenesis and fetal growth; (3) Trophoblast invasion of decidua and spiral arteries is driven by genes derived from both parents as well as by maternal nutritional factors; (4) Angiogenesis, vasculogenesis and vascular function are dependent on the genetic constitution of the embryo, derived from both parents, and the maternal genetically controlled nutritional environment.Early intra-uterine programming of vessels may concern the same (in)dependent determinants of vascular-related complications during pregnancy and cardiovascular diseases in later life.

Differential nuclear remodeling of mammalian somatic cells by Xenopus laevis oocyte and egg cytoplasm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alberio, Ramiro; Johnson, Andrew D.; Stick, Reimer

2005-07-01

The mechanisms governing nuclear reprogramming have not been fully elucidated yet; however, recent studies show a universally conserved ability of both oocyte and egg components to reprogram gene expression in somatic cells. The activation of genes associated with pluripotency by oocyte/egg components may require the remodeling of nuclear structures, such that they can acquire the features of early embryos and pluripotent cells. Here, we report on the remodeling of the nuclear lamina of mammalian cells by Xenopus oocyte and egg extracts. Lamin A/C is removed from somatic cells incubated in oocyte and egg extracts in an active process that requiresmore » permeable nuclear pores. Removal of lamin A/C is specific, since B-type lamins are not changed, and it is not dependent on the incorporation Xenopus egg specific lamin III. Moreover, transcriptional activity is differentially regulated in somatic cells incubated in the extracts. Pol I and II transcriptions are maintained in cells in oocyte extracts; however, both activities are abolished in egg extracts. Our study shows that components of oocyte and egg extracts can modify the nuclear lamina of somatic cells and that this nuclear remodeling induces a structural change in the nucleus which may have implications for transcriptional activity. These experiments suggest that modifications in the nuclear lamina structure by the removal of somatic proteins and the incorporation of oocyte/egg components may contribute to the reprogramming of somatic cell nuclei and may define a characteristic configuration of pluripotent cells.« less

Nasal heterotopia versus pilocytic astrocytoma: A narrow border.

PubMed

Ellouze, N; Born, J; Hoyoux, C; Michotte, A; Retz, C; Tebache, M; Piette, C

2015-08-01

Failure of the anterior neuropore can lead to three main types of anomalies: nasal dermal sinus, encephalocele and nasal glioma or heterotopia. In this report, we describe a case of intracranial and extracranial glial heterotopia that probably resulted from a common failure of anterior neuropore development. We describe the prenatal radiological assessment based on ultrasound and MRI results, and consider their limitation for early fetal diagnosis. We also discuss the embryogenesis and the possible pathogenic mechanisms involved. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Report of the NASA Mammalian Developmental Biology Working Group

NASA Technical Reports Server (NTRS)

Keefe, J. R.

1985-01-01

Development is considered to encompass all aspects of the mammalian life span from initial initial germ cell production through the complete life cycle to death of the organism. Thus, gamete production, fertilization, embryogenesis, implantation, fetogenesis, birth, peri- and postnatal maturation, and aging were all considered as stages of a development continuum relevant to problems of Space Biology. Deliberations thus far have been limited to stages of the development cycle from fertilization to early postnatal life. The deliberations are detailed.

Targeted deletion of p97 (VCP/CDC48) in mouse results in early embryonic lethality.

PubMed

Müller, J M M; Deinhardt, K; Rosewell, I; Warren, G; Shima, D T

2007-03-09

The highly conserved AAA ATPase p97 (VCP/CDC48) has well-established roles in cell cycle progression, proteasome degradation and membrane dynamics. Gene disruption in Saccromyces cerevisiae, Drosophila melanogaster and Trypanosoma brucei demonstrated that p97 is essential in unicellular and multicellular organisms. To explore the requirement for p97 in mammalian cell function and embryogenesis, we disrupted the p97 locus by gene targeting. Heterozygous p97+/- mice were indistinguishable from their wild-type littermates, whereas homozygous mutants did not survive to birth and died at a peri-implantation stage. These results show that p97 is an essential gene for early mouse development.

Cell death and morphogenesis during early mouse development: Are they interconnected?

PubMed Central

Bedzhov, Ivan; Zernicka-Goetz, Magdalena

2015-01-01

Shortly after implantation the embryonic lineage transforms from a coherent ball of cells into polarized cup shaped epithelium. Recently we elucidated a previously unknown apoptosis-independent morphogenic event that reorganizes the pluripotent lineage. Polarization cues from the surrounding basement membrane rearrange the epiblast into a polarized rosette-like structure, where subsequently a central lumen is established. Thus, we provided a new model revising the current concept of apoptosis-dependent epiblast morphogenesis. Cell death however has to be tightly regulated during embryogenesis to ensure developmental success. Here, we follow the stages of early mouse development and take a glimpse at the critical signaling and morphogenic events that determine cells destiny and reshape the embryonic lineage. PMID:25640415

Selection and validation of suitable reference genes for miRNA expression normalization by quantitative RT-PCR in citrus somatic embryogenic and adult tissues.

PubMed

Kou, Shu-Jun; Wu, Xiao-Meng; Liu, Zheng; Liu, Yuan-Long; Xu, Qiang; Guo, Wen-Wu

2012-12-01

miRNAs have recently been reported to modulate somatic embryogenesis (SE), a key pathway of plant regeneration in vitro. For expression level detection and subsequent function dissection of miRNAs in certain biological processes, qRT-PCR is one of the most effective and sensitive techniques, for which suitable reference gene selection is a prerequisite. In this study, three miRNAs and eight non-coding RNAs (ncRNA) were selected as reference candidates, and their expression stability was inspected in developing citrus SE tissues cultured at 20, 25, and 30 °C. Stability of the eight non-miRNA ncRNAs was further validated in five adult tissues without temperature treatment. The best single reference gene for SE tissues was snoR14 or snoRD25, while for the adult tissues the best one was U4; although they were not as stable as the optimal multiple references snoR14 + U6 for SE tissues and snoR14 + U5 for adult tissues. For expression normalization of less abundant miRNAs in SE tissues, miR3954 was assessed as a viable reference. Single reference gene snoR14 outperformed multiple references for the overall SE and adult tissues. As one of the pioneer systematic studies on reference gene identification for plant miRNA normalization, this study benefits future exploration on miRNA function in citrus and provides valuable information for similar studies in other higher plants. Three miRNAs and eight non-coding RNAs were tested as reference candidates on developing citrus SE tissues. Best single references snoR14 or snoRD25 and optimal multiple references snoR14 + U6, snoR14 + U5 were identified.

Norway spruce embryogenesis: changes in carbohydrate profile, structural development and response to polyethylene glycol

PubMed Central

Hudec, Lukáš; Konrádová, Hana; Hašková, Anna; Lipavská, Helena

2016-01-01

Two unrelated, geographically distinct, highly embryogenic lines of Norway spruce (Picea abies (L.) Karst.) were analysed to identify metabolic traits characteristic for lines with good yields of high-quality embryos. The results were compared with corresponding characteristics of a poorly productive line (low embryo yield, scarce high-quality embryos). The following carbohydrate profiles and spectra during maturation, desiccation and germination were identified as promising characteristics for line evaluation: a gradual decrease in total soluble carbohydrates with an increasing sucrose : hexose ratio during maturation; accumulation of raffinose family oligosaccharides resulting from desiccation and their rapid degradation at the start of germination; and a decrease in sucrose, increase in hexoses and the appearance of pinitol with proceeding germination. We propose that any deviation from this profile in an embryonic line is a symptom of inferior somatic embryo development. We further propose that a fatty acid spectrum dominated by linoleic acid (18 : 2) was a common feature of healthy spruce somatic embryos, although it was quite different from zygotic embryos mainly containing oleic acid (18 : 1). The responses of the lines to osmotic stress were evaluated based on comparison of control (without osmoticum) and polyethylene glycol (PEG)-exposed (PEG 4000) variants. Although genetically distinct, both highly embryogenic lines responded in a very similar manner, with the only difference being sensitivity to high concentrations of PEG. At an optimum PEG concentration (3.75 and 5%), which was line specific, negative effects of PEG on embryo germination were compensated for by a higher maturation efficiency so that the application of PEG at an appropriate concentration improved the yield of healthy germinants per gram of initial embryonal mass and accelerated the process. Polyethylene glycol application, however, resulted in no improvement of the poorly productive line. PMID:27052433

Tissue culture and associated biotechnological interventions for the improvement of coconut (Cocos nucifera L.): a review.

PubMed

Nguyen, Quang Thien; Bandupriya, H D Dharshani; López-Villalobos, Arturo; Sisunandar, S; Foale, Mike; Adkins, Steve W

2015-11-01

The present review discusses not only advances in coconut tissue culture and associated biotechnological interventions but also future research directions toward the resilience of this important palm crop. Coconut (Cocos nucifera L.) is commonly known as the 'tree of life'. Every component of the palm can be used to produce items of value and many can be converted into industrial products. Coconut cultivation faces a number of acute problems that reduce its productivity and competitiveness. These problems include various biotic and abiotic challenges as well as an unstable market for its traditional oil-based products. Around 10 million small-holder farmers cultivate coconut palms worldwide on c. 12 million hectares of land, and many more people own a few coconut palms that contribute to their livelihoods. Inefficiency in the production of seedlings for replanting remains an issue; however, tissue culture and other biotechnological interventions are expected to provide pragmatic solutions. Over the past 60 years, much research has been directed towards developing and improving protocols for (i) embryo culture; (ii) clonal propagation via somatic embryogenesis; (iii) homozygote production via anther culture; (iv) germplasm conservation via cryopreservation; and (v) genetic transformation. Recently other advances have revealed possible new ways to improve these protocols. Although effective embryo culture and cryopreservation are now possible, the limited frequency of conversion of somatic embryos to ex vitro seedlings still prevents the large-scale clonal propagation of coconut. This review illustrates how our knowledge of tissue culture and associated biotechnological interventions in coconut has so far developed. Further improvement of protocols and their application to a wider range of germplasm will continue to open up new horizons for the collection, conservation, breeding and productivity of coconut.

Non-staining visualization of embryogenesis and energy metabolism in medaka fish eggs using near-infrared spectroscopy and imaging.

PubMed

Puangchit, Paralee; Ishigaki, Mika; Yasui, Yui; Kajita, Misato; Ritthiruangdej, Pitiporn; Ozaki, Yukihiro

2017-12-04

The energy metabolism and embryogenesis of fertilized Japanese medaka eggs were investigated in vivo at the molecular level using near-infrared (NIR) spectroscopy and imaging. Changes in chemical components, such as proteins and lipids, in yolk sphere and embryonic body were studied over the course of embryonic development. Metabolic changes that represent variations in the concentrations and molecular compositions of proteins and lipids in the yolk part, particularly on the 1 st day after fertilization and the day just before hatching, were successfully identified in the 4900-4000 cm -1 wavenumber region. The yolk components were shown to have specific functions at the very early and final stages of the embryonic development. Proteins with α-helix- or β-sheet-rich structures clearly showed the different variation patterns within the developing egg. Furthermore, the distribution of lipids could be selectively visualized using data from the higher wavenumber region. Detailed embryonic structures were clearly depicted in the NIR images using the data from the 6400-5500 cm -1 region in which the embryo parts had some characteristic peaks due to unsaturated fatty acids. It was made clear that yolk and embryo parts had different components especially lipid components. The present study provides new insights into material variations in the fertilized egg during its growth. NIR imaging proved to be valuable in investigating the embryogenesis in vivo at the molecular level in terms of changes in biomolecular concentrations and compositions, metabolic differentiation, and detailed information about embryonic structures without the need for staining.

Hedgehog signaling is required for formation of the notochord sheath and patterning of nuclei pulposi within the intervertebral discs

PubMed Central

Choi, Kyung-Suk; Harfe, Brian D.

2011-01-01

The vertebrae notochord is a transient rod-like structure that produces secreted factors that are responsible for patterning surrounding tissues. During later mouse embryogenesis, the notochord gives rise to the middle part of the intervertebral disc, called the nucleus pulposus. Currently, very little is known about the molecular mechanisms responsible for forming the intervertebral discs. Here we demonstrate that hedgehog signaling is required for formation of the intervertebral discs. Removal of hedgehog signaling in the notochord and nearby floorplate resulted in the formation of an aberrant notochord sheath that normally surrounds this structure. In the absence of the notochord sheath, small nuclei pulposi were formed, with most notochord cells dispersed throughout the vertebral bodies during embryogenesis. Our data suggest that the formation of the notochord sheath requires hedgehog signaling and that the sheath is essential for maintaining the rod-like structure of the notochord during early embryonic development. As notochord cells form nuclei pulposi, we propose that the notochord sheath functions as a “wrapper” around the notochord to constrain these cells along the vertebral column. PMID:21606373

Effect of phenol on embryo development and expression of metallothionein in the sea urchin Hemicentrotus pulcherrimus

NASA Astrophysics Data System (ADS)

Hwang, Un-Ki; Lee, Ju-Wook; Ryu, Hyang-Mi; Kang, Ju-Chan; Kang, Han Seung

2015-12-01

In this study, we identified and cloned the sea urchin Hemicentrotus pulcherrimus MT (Hp-MT) mRNA. We examined the gameto- and embryo-toxic effects and the expression of Hp-MT mRNA at various concentrations of phenol in H. pulcherrimus. We found that the normal embryogenesis rate was significantly inhibited when H. pulcherrimus was exposed to phenol (EC50 = 1565.86 ppb, 95% Cl = 1183.47-2037.84 ppb). The no observed effective concentration (NOEC) and the lowest observed effective concentration (LOEC) of the normal embryogenesis rate were < 10 ppb and 100 ppb, respectively. Hp-MT cDNA is 651 bp in length and encodes a protein of 64 amino acids. We found that the expression of Hp-MT mRNA was significantly increased with phenol treatment in a concentrationdependent manner. These results suggest that phenol at greater than 100 ppb has a toxic effect during the early embryonic stages of H. pulcherrimus, and MT mRNA may be used as a biomarker for risk assessment of phenol contamination.

Hedgehog signaling is required for formation of the notochord sheath and patterning of nuclei pulposi within the intervertebral discs.

PubMed

Choi, Kyung-Suk; Harfe, Brian D

2011-06-07

The vertebrae notochord is a transient rod-like structure that produces secreted factors that are responsible for patterning surrounding tissues. During later mouse embryogenesis, the notochord gives rise to the middle part of the intervertebral disc, called the nucleus pulposus. Currently, very little is known about the molecular mechanisms responsible for forming the intervertebral discs. Here we demonstrate that hedgehog signaling is required for formation of the intervertebral discs. Removal of hedgehog signaling in the notochord and nearby floorplate resulted in the formation of an aberrant notochord sheath that normally surrounds this structure. In the absence of the notochord sheath, small nuclei pulposi were formed, with most notochord cells dispersed throughout the vertebral bodies during embryogenesis. Our data suggest that the formation of the notochord sheath requires hedgehog signaling and that the sheath is essential for maintaining the rod-like structure of the notochord during early embryonic development. As notochord cells form nuclei pulposi, we propose that the notochord sheath functions as a "wrapper" around the notochord to constrain these cells along the vertebral column.

Pollen embryogenesis to induce, detect, and analyze mutants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Constantin, M.J.

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 tomore » 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.« less

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

2015-09-04

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.

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

PubMed Central

Di Pasquale, Elisa; Brivanlou, Ali H.

2009-01-01

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

Transient Exposure to Ethanol during Zebrafish Embryogenesis Results in Defects in Neuronal Differentiation: An Alternative Model System to Study FASD

PubMed Central

Joya, Xavier; Garcia-Algar, Oscar; Vall, Oriol; Pujades, Cristina

2014-01-01

Background The exposure of the human embryo to ethanol results in a spectrum of disorders involving multiple organ systems, including the impairment of the development of the central nervous system (CNS). In spite of the importance for human health, the molecular basis of prenatal ethanol exposure remains poorly understood, mainly to the difficulty of sample collection. Zebrafish is now emerging as a powerful organism for the modeling and the study of human diseases. In this work, we have assessed the sensitivity of specific subsets of neurons to ethanol exposure during embryogenesis and we have visualized the sensitive embryonic developmental periods for specific neuronal groups by the use of different transgenic zebrafish lines. Methodology/Principal Findings In order to evaluate the teratogenic effects of acute ethanol exposure, we exposed zebrafish embryos to ethanol in a given time window and analyzed the effects in neurogenesis, neuronal differentiation and brain patterning. Zebrafish larvae exposed to ethanol displayed small eyes and/or a reduction of the body length, phenotypical features similar to the observed in children with prenatal exposure to ethanol. When neuronal populations were analyzed, we observed a clear reduction in the number of differentiated neurons in the spinal cord upon ethanol exposure. There was a decrease in the population of sensory neurons mainly due to a decrease in cell proliferation and subsequent apoptosis during neuronal differentiation, with no effect in motoneuron specification. Conclusion Our investigation highlights that transient exposure to ethanol during early embryonic development affects neuronal differentiation although does not result in defects in early neurogenesis. These results establish the use of zebrafish embryos as an alternative research model to elucidate the molecular mechanism(s) of ethanol-induced developmental toxicity at very early stages of embryonic development. PMID:25383948

Selective loss of mouse embryos due to the expression of transgenic major histocompatibility class I molecules early in embryogenesis.

PubMed

Aït-Azzouzene, D; Langkopf, A; Cohen, J; Bleux, C; Gendron, M C; Kanellopoulos-Langevin, C

1998-05-01

Among the numerous hypotheses proposed to explain the absence of fetal rejection by the mother in mammals, it has been suggested that regulation of expression of the polymorphic major histocompatibility complex (MHC) at the fetal-maternal interface plays a major role. In addition to a lack of MHC gene expression in the placenta throughout gestation, the absence of polymorphic MHC molecules on the early embryo, as well as their low level of expression after midgestation, could contribute to this important biologic phenomenon. In order to test this hypothesis, we have produced transgenic mice able to express polymorphic MHC class I molecules early in embryogenesis. We have placed the MHC class la gene H-2Kb under the control of a housekeeping gene promoter, the hydroxy-methyl-glutaryl coenzyme A reductase (HMG) gene minimal promoter. This construct has been tested for functionality after transfection into mouse fibroblast L cells. The analysis of three founder transgenic mice and their progeny suggested that fetoplacental units that could express the H-2Kb heavy chains are unable to survive in utero beyond midgestation. We have shown further that a much higher resorption rate, on days 11 to 13 of embryonic development, is observed among transgenic embryos developing from eggs microinjected at the one-cell stage with the pHMG-Kb construct than in control embryos. This lethality is not due to immune phenomena, since it is observed in histocompatible combinations between mother and fetus. These results are discussed in the context of what is currently known about the regulation of MHC expression at the fetal-maternal interface and in various transgenic mouse models.

Changes in ecdysteroid levels and expression patterns of ecdysteroid-responsive factors and neuropeptide hormones during the embryogenesis of the blue crab, Callinectes sapidus.

PubMed

Techa, Sirinart; Alvarez, Javier V; Sook Chung, J

2015-04-01

Embryogenesis requires the involvement and coordination of multiple networks of various genes, according to a timeline governing development. Crustacean embryogenesis usually includes the first molt, a process that is known to be positively controlled by ecdysteroids. We determined the amounts of ecdysteroids, as well as other related factors: the ecdysone receptor (CasEcR), the retinoid X receptor (CasRXR), the molt-inhibiting hormone (CasMIH), and crustacean hyperglycemic hormone (CasCHH) during the ovarian and embryonic developments of Callinectes sapidus. In summary, the ovaries at stages 1-4 have expression levels of maternal CasEcR and CasRXR 10-50 times higher than levels seen in embryos at the yolk stage. This large difference in the amount of the these factors in C. sapidus ovaries suggests that these maternal ecdysteroid-responsive factors may be utilized at the initiation of embryogenesis. During embryogenesis, the changes in total ecdysteroids and levels of CasEcR and CasRXR expression are similar to those observed in juvenile molts. The full-length cDNA sequence of the C. sapidus BTB domain protein (CasBTBDP) initially isolated from Y-organ cDNA, contains only Broad-Complex, Tramtrack, and Bric a brac (BTB) domains. The levels of CasBTBDP are kept constant throughout embryogenesis. The expression profiles of CasMIH and CasCHH are similar to the titers of ecdysteroids. However, the timing of their appearance is followed by increases in CasEcRs and CasRXRs, implying that the expressions of these neuropeptides may be influenced by ecdysteroids. Moreover, the ecdysteroid profile during embryogenesis may track directly with the timing of organogenesis of Y-organs and their activity. Our work reports, for first time, the observed expression and changes of ecdysteroid-responsive factors, along with CasCHH and CasMIH, during embryogenesis in the crustacean C. sapidus. Copyright © 2014 Elsevier Inc. All rights reserved.

Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture.

PubMed

Chan, Pek-Lan; Rose, Ray J; Abdul Murad, Abdul Munir; Zainal, Zamri; Low, Eng-Ti Leslie; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

2014-01-01

The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate reference genes in other oil palm tissues and in the expression profiling of genes relating to yield, biotic and abiotic stresses.

The developmental basis for germline mosaicism in mouse and Drosophila melanogaster.

PubMed

Drost, J B; Lee, W R

1998-01-01

Data involving germline mosaics in Drosophila melanogaster and mouse are reconciled with developmental observations. Mutations that become fixed in the early embryo before separation of soma from the germline may, by the sampling process of development, continue as part of germline and/or differentiate into any somatic tissue. The cuticle of adult D. melanogaster, because of segmental development, can be used to estimate the proportion of mutant nuclei in the early embryo, but most somatic tissues and the germlines of both species continue from samples too small to be representative of the early embryo. Because of the small sample of cells/nuclei that remain in the germline after separation of soma in both species, mosaic germlines have percentages of mutant cells that vary widely, with a mean of 50% and an unusual platykurtic, flat-topped distribution. While the sampling process leads to similar statistical results for both species, their patterns of development are very different. In D. melanogaster the first differentiation is the separation of soma from germline with the germline continuing from a sample of only two to four nuclei, whereas the adult cuticle is a representative sample of cleavage nuclei. The presence of mosaicism in D. melanogaster germline is independent of mosaicism in the eye, head, and thorax. This independence was used to determine that mutations can occur at any of the early embryonic cell divisions and still average 50% mutant germ cells when the germline is mosaic; however, the later the mutation occurs, the higher the proportion of completely nonmutant germlines. In contrast to D. melanogaster, the first differentiation in the mouse does not separate soma from germline but produces the inner cell mass that is representative of the cleavage nuclei. Following formation of the primitive streak, the primordial germ cells develop at the base of the allantois and among a clonally related sample of cells, providing the same statistical distribution in the mouse germlines as in D. melanogaster. The proportion of mutations that are fixed during early embryonic development is greatly underestimated. For example, a DNA lesion in a postmeiotic gamete that becomes fixed as a dominant mutation during early embryonic development of the F1 may produce an individual completely mutant in the germ line and relevant somatic tissue or, alternatively, the F1 germline may be completely mutant but with no relevant somatic tissues for detecting the mutation until the F2. In both cases the mutation would be classified as complete in the F1 and F2, respectively, and not recognized as embryonic in origin. Because germ cells differentiate later in mammalian development, there are more opportunities for correlation between germline and soma in the mammal than Drosophila. However, because the germ cells and any somatic tissue, like blood, are derived from small samples, there may be many individuals that test negative in blood but have germlines that are either mosaic or entirely mutant.

ADAM13 Induces Cranial Neural Crest by Cleaving Class B Ephrins and Regulating Wnt Signaling

PubMed Central

Wei, Shuo; Xu, Guofeng; Bridges, Lance C.; Williams, Phoebe; White, Judith M.; DeSimone, Douglas W.

2010-01-01

SUMMARY The cranial neural crest (CNC) are multipotent embryonic cells that contribute to craniofacial structures and other cells and tissues of the vertebrate head. During embryogenesis, CNC is induced at the neural plate boundary through the interplay of several major signaling pathways. Here we report that the metalloproteinase activity of ADAM13 is required for early induction of CNC in Xenopus. In both cultured cells and X. tropicalis embryos, membrane-bound Ephrins (Efns) B1 and B2 were identified as substrates for ADAM13. ADAM13 upregulates canonical Wnt signaling and early expression of the transcription factor snail2, whereas EfnB1 inhibits the canonical Wnt pathway and snail2 expression. We propose that by cleaving class B Efns, ADAM13 promotes canonical Wnt signaling and early CNC induction. PMID:20708595

Central cell-derived peptides regulate early embryo patterning in flowering plants.

PubMed

Costa, Liliana M; Marshall, Eleanor; Tesfaye, Mesfin; Silverstein, Kevin A T; Mori, Masashi; Umetsu, Yoshitaka; Otterbach, Sophie L; Papareddy, Ranjith; Dickinson, Hugh G; Boutiller, Kim; VandenBosch, Kathryn A; Ohki, Shinya; Gutierrez-Marcos, José F

2014-04-11

Plant embryogenesis initiates with the establishment of an apical-basal axis; however, the molecular mechanisms accompanying this early event remain unclear. Here, we show that a small cysteine-rich peptide family is required for formation of the zygotic basal cell lineage and proembryo patterning in Arabidopsis. EMBRYO SURROUNDING FACTOR 1 (ESF1) peptides accumulate before fertilization in central cell gametes and thereafter in embryo-surrounding endosperm cells. Biochemical and structural analyses revealed cleavage of ESF1 propeptides to form biologically active mature peptides. Further, these peptides act in a non-cell-autonomous manner and synergistically with the receptor-like kinase SHORT SUSPENSOR to promote suspensor elongation through the YODA mitogen-activated protein kinase pathway. Our findings demonstrate that the second female gamete and its sexually derived endosperm regulate early embryonic patterning in flowering plants.

Perspectives and Open Problems in the Early Phases of Left-Right Patterning

PubMed Central

Vandenberg, Laura N.; Levin, Michael

2009-01-01

Summary Embryonic left-right (LR) patterning is a fascinating aspect of embryogenesis. The field currently faces important questions about the origin of LR asymmetry, the mechanisms by which consistent asymmetry is imposed on the scale of the whole embryo, and the degree of conservation of early phases of LR patterning among model systems. Recent progress on planar cell polarity and cellular asymmetry in a variety of tissues and species provides a new perspective on the early phases of LR patterning. Despite the huge diversity in body-plans over which consistent LR asymmetry is imposed, and the apparent divergence in molecular pathways that underlie laterality, the data reveal conservation of physiological modules among phyla and a basic scheme of cellular chirality amplified by a planar cell polarity-like pathway over large cell fields. PMID:19084609

FGF8 coordinates tissue elongation and cell epithelialization during early kidney tubulogenesis

PubMed Central

Atsuta, Yuji; Takahashi, Yoshiko

2015-01-01

When a tubular structure forms during early embryogenesis, tubular elongation and lumen formation (epithelialization) proceed simultaneously in a spatiotemporally coordinated manner. We here demonstrate, using the Wolffian duct (WD) of early chicken embryos, that this coordination is regulated by the expression of FGF8, which shifts posteriorly during body axis elongation. FGF8 acts as a chemoattractant on the leader cells of the elongating WD and prevents them from epithelialization, whereas static (‘rear’) cells that receive progressively less FGF8 undergo epithelialization to form a lumen. Thus, FGF8 acts as a binary switch that distinguishes tubular elongation from lumen formation. The posteriorly shifting FGF8 is also known to regulate somite segmentation, suggesting that multiple types of tissue morphogenesis are coordinately regulated by macroscopic changes in body growth. PMID:26130757

CENH3-GFP: a visual marker for gametophytic and somatic ploidy determination in Arabidopsis thaliana.

PubMed

De Storme, Nico; Keçeli, Burcu Nur; Zamariola, Linda; Angenon, Geert; Geelen, Danny

2016-01-05

The in vivo determination of the cell-specific chromosome number provides a valuable tool in several aspects of plant research. However, current techniques to determine the endosystemic ploidy level do not allow non-destructive, cell-specific chromosome quantification. Particularly in the gametophytic cell lineages, which are physically encapsulated in the reproductive organ structures, direct in vivo ploidy determination has been proven very challenging. Using Arabidopsis thaliana as a model, we here assess the applicability of recombinant CENH3-GFP reporters for the labeling of the cell's chromocenters and for the monitoring of the gametophytic and somatic chromosome number in vivo. By modulating expression of a CENH3-GFP reporter cassette using different promoters, we isolated two reporter lines that allow for a clear and highly specific labeling of centromeric chromosome regions in somatic and gametophytic cells respectively. Using polyploid plant series and reproductive mutants, we demonstrate that the pWOX2-CENH3-GFP recombinant fusion protein allows for the determination of the gametophytic chromosome number in both male and female gametophytic cells, and additionally labels centromeric regions in early embryo development. Somatic centromere labeling through p35S-CENH3-GFP shows a maximum of ten centromeric dots in young dividing tissues, reflecting the diploid chromosome number (2x = 10), and reveals a progressive decrease in GFP foci frequency throughout plant development. Moreover, using chemical and genetic induction of endomitosis, we demonstrate that CENH3-mediated chromosome labeling provides an easy and valuable tool for the detection and characterization of endomitotic polyploidization events. This study demonstrates that the introgression of the pWOX2-CENH3-GFP reporter construct in Arabidopsis thaliana provides an easy and reliable methodology for determining the chromosome number in developing male and female gametes, and during early embryo development. Somatically expressed CENH3-GFP reporters, on the other hand, constitute a valuable tool to quickly determine the basic somatic ploidy level in young seedlings at the individual cell level and to detect and to quantify endomitotic polyploidization events in a non-destructive, microscopy-based manner.

Identification and profiling of conserved and novel microRNAs involved in oil and oleic acid production during embryogenesis in Carya cathayensis Sarg.

PubMed

Wang, Zhengjia; Huang, Ruiming; Sun, Zhichao; Zhang, Tong; Huang, Jianqin

2017-05-01

MicroRNAs (miRNAs) are important regulators of plant development and fruit formation. Mature embryos of hickory (Carya cathayensis Sarg.) nuts contain more than 70% oil (comprising 90% unsaturated fatty acids), along with a substantial amount of oleic acid. To understand the roles of miRNAs involved in oil and oleic acid production during hickory embryogenesis, three small RNA libraries from different stages of embryogenesis were constructed. Deep sequencing of these three libraries identified 95 conserved miRNAs with 19 miRNA*s, 7 novel miRNAs (as well as their corresponding miRNA*s), and 26 potentially novel miRNAs. The analysis identified 15 miRNAs involved in oil and oleic acid production that are differentially expressed during embryogenesis in hickory. Among them, nine miRNA sequences, including eight conserved and one novel, were confirmed by qRT-PCR. In addition, 145 target genes of the novel miRNAs were predicted using a bioinformatic approach. Our results provide a framework for better understanding the roles of miRNAs during embryogenesis in hickory.

Genetics Home Reference: primary myelofibrosis

MedlinePlus

... from gene mutations that occur in early blood-forming cells after conception. These alterations are called somatic ... Free article on PubMed Central Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD, ...

Early improvement with pregabalin predicts endpoint response in patients with generalized anxiety disorder: an integrated and predictive data analysis.

PubMed

Montgomery, Stuart A; Lyndon, Gavin; Almas, Mary; Whalen, Ed; Prieto, Rita

2017-01-01

Generalized anxiety disorder (GAD), a common mental disorder, has several treatment options including pregabalin. Not all patients respond to treatment; quickly determining which patients will respond is an important treatment goal. Patient-level data were pooled from nine phase II and III randomized, double-blind, short-term, placebo-controlled trials of pregabalin for the treatment of GAD. Efficacy outcomes included the change from baseline in the Hamilton Anxiety Scale (HAM-A) total score and psychic and somatic subscales. Predictive modelling assessed baseline characteristics and early clinical responses to determine those predictive of clinical improvement at endpoint. A total of 2155 patients were included in the analysis (1447 pregabalin, 708 placebo). Pregabalin significantly improved the HAM-A total score compared with the placebo at endpoint, treatment difference (95% confidence interval), -2.61 (-3.21 to -2.01), P<0.0001. Pregabalin significantly improved HAM-A psychic and somatic scores compared with placebo, -1.52 (-1.85 to -1.18), P<0.0001, and -1.10 (-1.41 to -0.80), P<0.0001, respectively. Response to pregabalin in the first 1-2 weeks (≥20 or ≥30% improvement in HAM-A total, psychic or somatic score) was predictive of an endpoint greater than or equal to 50% improvement in the HAM-A total score. Pregabalin is an effective treatment option for patients with GAD. Patients with early response to pregabalin are more likely to respond significantly at endpoint.

Somatic POLE exonuclease domain mutations are early events in sporadic endometrial and colorectal carcinogenesis, determining driver mutational landscape, clonal neoantigen burden and immune response.

PubMed

Temko, Daniel; Van Gool, Inge C; Rayner, Emily; Glaire, Mark; Makino, Seiko; Brown, Matthew; Chegwidden, Laura; Palles, Claire; Depreeuw, Jeroen; Beggs, Andrew; Stathopoulou, Chaido; Mason, John; Baker, Ann-Marie; Williams, Marc; Cerundolo, Vincenzo; Rei, Margarida; Taylor, Jenny C; Schuh, Anna; Ahmed, Ahmed; Amant, Frédéric; Lambrechts, Diether; Smit, Vincent Thbm; Bosse, Tjalling; Graham, Trevor A; Church, David N; Tomlinson, Ian

2018-03-31

Genomic instability, which is a hallmark of cancer, is generally thought to occur in the middle to late stages of tumourigenesis, following the acquisition of permissive molecular aberrations such as TP53 mutation or whole genome doubling. Tumours with somatic POLE exonuclease domain mutations are notable for their extreme genomic instability (their mutation burden is among the highest in human cancer), distinct mutational signature, lymphocytic infiltrate, and excellent prognosis. To what extent these characteristics are determined by the timing of POLE mutations in oncogenesis is unknown. Here, we have shown that pathogenic POLE mutations are detectable in non-malignant precursors of endometrial and colorectal cancer. Using genome and exome sequencing, we found that multiple driver mutations in POLE-mutant cancers show the characteristic POLE mutational signature, including those in genes conventionally regarded as initiators of tumourigenesis. In POLE-mutant cancers, the proportion of monoclonal predicted neoantigens was similar to that in other cancers, but the absolute number was much greater. We also found that the prominent CD8 + T-cell infiltrate present in POLE-mutant cancers was evident in their precursor lesions. Collectively, these data indicate that somatic POLE mutations are early, quite possibly initiating, events in the endometrial and colorectal cancers in which they occur. The resulting early onset of genomic instability may account for the striking immune response and excellent prognosis of these tumours, as well as their early presentation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

A protein domain-based interactome network for C. elegans early embryogenesis

PubMed Central

Boxem, Mike; Maliga, Zoltan; Klitgord, Niels; Li, Na; Lemmens, Irma; Mana, Miyeko; de Lichtervelde, Lorenzo; Mul, Joram D.; van de Peut, Diederik; Devos, Maxime; Simonis, Nicolas; Yildirim, Muhammed A.; Cokol, Murat; Kao, Huey-Ling; de Smet, Anne-Sophie; Wang, Haidong; Schlaitz, Anne-Lore; Hao, Tong; Milstein, Stuart; Fan, Changyu; Tipsword, Mike; Drew, Kevin; Galli, Matilde; Rhrissorrakrai, Kahn; Drechsel, David; Koller, Daphne; Roth, Frederick P.; Iakoucheva, Lilia M.; Dunker, A. Keith; Bonneau, Richard; Gunsalus, Kristin C.; Hill, David E.; Piano, Fabio; Tavernier, Jan; van den Heuvel, Sander; Hyman, Anthony A.; Vidal, Marc

2008-01-01

Summary Many protein-protein interactions are mediated through independently folding modular domains. Proteome-wide efforts to model protein-protein interaction or “interactome” networks have largely ignored this modular organization of proteins. We developed an experimental strategy to efficiently identify interaction domains and generated a domain-based interactome network for proteins involved in C. elegans early embryonic cell divisions. Minimal interacting regions were identified for over 200 proteins, providing important information on their domain organization. Furthermore, our approach increased the sensitivity of the two-hybrid system, resulting in a more complete interactome network. This interactome modeling strategy revealed new insights into C. elegans centrosome function and is applicable to other biological processes in this and other organisms. PMID:18692475

Cell death and morphogenesis during early mouse development: are they interconnected?

PubMed

Bedzhov, Ivan; Zernicka-Goetz, Magdalena

2015-04-01

Shortly after implantation the embryonic lineage transforms from a coherent ball of cells into polarized cup shaped epithelium. Recently we elucidated a previously unknown apoptosis-independent morphogenic event that reorganizes the pluripotent lineage. Polarization cues from the surrounding basement membrane rearrange the epiblast into a polarized rosette-like structure, where subsequently a central lumen is established. Thus, we provided a new model revising the current concept of apoptosis-dependent epiblast morphogenesis. Cell death however has to be tightly regulated during embryogenesis to ensure developmental success. Here, we follow the stages of early mouse development and take a glimpse at the critical signaling and morphogenic events that determine cells destiny and reshape the embryonic lineage. © 2015 The Authors. Bioessays published by WILEY Periodicals, Inc.

[Specification of cell destiny in early Caenorhabditis elegans embryo].

PubMed

Schierenberg, E

1997-02-01

Embryogenesis of the nematode Caenorhabditis elegans has been described completely on a cell-by-cell basis and found to be essentially invariant. With this knowledge in hands, micromanipulated embryos and mutants have been analyzed for cell lineage defects and the distribution of specific gene products. The results challenge the classical view of cell-autonomous development in nematodes and indicate that the early embryo of C. elegans is a highly dynamic system. A network of inductive events between neighboring cells is being revealed, which is necessary to assign different developmental programs to blastomeres. In those cases where molecules involved in these cell-cell interactions have been identified, homologies to cell surface receptors, ligands and transcription factors found in other systems have become obvious.

Metabolic circadian rhythms in embryonic turtles.

PubMed

Loudon, Fiona Kay; Spencer, Ricky-John; Strassmeyer, Alana; Harland, Karen

2013-07-01

Oviparous species are model organisms for investigating embryonic development of endogenous physiological circadian rhythms without the influence of maternal biorhythms. Recent studies have demonstrated that heart rates and metabolic rates of embryonic turtles are not constant or always maximal and can be altered in response to the presence of embryos at a more advanced stage of development within the nest. A first step in understanding the physiological mechanisms underpinning these responses in embryonic ectothermic organisms is to develop metabolic profiles (e.g., heart rate) at different temperatures throughout incubation. Heart beat and rhythmic patterns or changes in development may represent important signals or cues within a nest and may be vital to coordinate synchronous hatching well in advance of the final stages of incubation. We developed baseline embryonic heart-rate profiles of embryos of the short-necked Murray River turtle (Emydura macquarii) to determine the stage of embryogenesis that metabolic circadian rhythms become established, if at all. Eggs were incubated at constant temperatures (26°C and 30°C) and heart rates were monitored at 6-h intervals over 24 h every 7-11 days until hatching. Circadian heart rate rhythms were detected at the mid-gestation period and were maintained until hatching. Heart rates throughout the day varied by up to 20% over 24 h and were not related to time of day. This study demonstrated that endogenous metabolic circadian rhythms in developing embryos in turtle eggs establish earlier in embryogenesis than those documented in other vertebrate taxa during embryogenesis. Early establishment of circadian rhythms in heart rates may be critical for communication among embryos and synchrony in hatching and emergence from the nest.

Copb2 is essential for embryogenesis and hypomorphic mutations cause human microcephaly.

PubMed

DiStasio, Andrew; Driver, Ashley; Sund, Kristen; Donlin, Milene; Muraleedharan, Ranjith M; Pooya, Shabnam; Kline-Fath, Beth; Kaufman, Kenneth M; Prows, Cynthia A; Schorry, Elizabeth; Dasgupta, Biplab; Stottmann, Rolf W

2017-12-15

Primary microcephaly is a congenital brain malformation characterized by a head circumference less than three standard deviations below the mean for age and sex and results in moderate to severe mental deficiencies and decreased lifespan. We recently studied two children with primary microcephaly in an otherwise unaffected family. Exome sequencing identified an autosomal recessive mutation leading to an amino acid substitution in a WD40 domain of the highly conserved Coatomer Protein Complex, Subunit Beta 2 (COPB2). To study the role of Copb2 in neural development, we utilized genome-editing technology to generate an allelic series in the mouse. Two independent null alleles revealed that Copb2 is essential for early stages of embryogenesis. Mice homozygous for the patient variant (Copb2R254C/R254C) appear to have a grossly normal phenotype, likely due to differences in corticogenesis between the two species. Strikingly, mice heterozygous for the patient mutation and a null allele (Copb2R254C/Zfn) show a severe perinatal phenotype including low neonatal weight, significantly increased apoptosis in the brain, and death within the first week of life. Immunostaining of the Copb2R254C/Zfnbrain revealed a reduction in layer V (CTIP2+) neurons, while the overall cell density of the cortex is unchanged. Moreover, neurospheres derived from animals with Copb2 variants grew less than control. These results identify a general requirement for COPB2 in embryogenesis and a specific role in corticogenesis. We further demonstrate the utility of CRISPR-Cas9 generated mouse models in the study of potential pathogenicity of variants of potential clinical interest. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Nanos3 not nanos1 and nanos2 is a germ cell marker gene in large yellow croaker during embryogenesis.

PubMed

Han, Kunhuang; Chen, Shihai; Cai, Mingyi; Jiang, Yonghua; Zhang, Ziping; Wang, Yilei

2018-04-01

In this study, three nanos gene subtypes (Lcnanos1, Lcnanos2 and Lcnanos3) from Larimichthys crocea, were cloned and characterized. We determined the spatio-temporal expression patterns of each subtype in tissues as well as the cellular localization of mRNA in embryos. Results showed that deduced Nanos proteins have two main homology domains: N-terminal CCR4/NOT1 deadenylase interaction domain and highly conserved carboxy-terminal region bearing two conserved CCHC zinc-finger motifs. The expression levels of Lcnanos1 in testis were significantly higher than other tissues, followed by heart, brain, eye, and ovary. Nevertheless, both Lcnanos2 and Lcnanos3 were restrictedly expressed in testis and ovary, respectively. No signals of Lcnanos1 and Lcnanos2 expression were detected at any developmental stages during embryogenesis. On the contrary, the signals of Lcnanos3 were detected in all stages examined. Lcnanos3 transcripts were firstly localized to the distal end of cleavage furrow at the 2-cell stage. Subsequently, mounting positive signals started to appear in a small number of cells as the embryo developed to blastula stage and early-gastrula stage. As development proceeded, positive signals were found in the primitive gonadal ridge. These cells of Lcnanos3 positive signals implied the specification of the future PGCs at this stage. It also suggested that PGCs of croaker originate from four clusters of cells which inherit maternal germ plasm at blastula stage. Furthermore, we preliminarily analyzed the migration route of PGCs in embryos of L. crocea. In short, this study laid the foundation for studies on specification and development of germ cell from L. crocea during embryogenesis. Copyright © 2018 Elsevier Inc. All rights reserved.

Sea Urchin Embryogenesis as Bioindicators of Marine Pollution in Impact Areas of the Sea of Japan/East Sea and the Sea of Okhotsk.

PubMed

Lukyanova, Olga N; Zhuravel, Elena V; Chulchekov, Denis N; Mazur, Andrey A

2017-08-01

The embryogenesis of the sea urchin sand dollar Scaphechinus mirabilis was used as bioindicators of seawater quality from the impact areas of the Sea of Japan/East Sea (Peter the Great Bay) and the Sea of Okhotsk (northwestern shelf of Sakhalin Island and western shelf of Kamchatka Peninsula). Fertilization membrane formation, first cleavage, blastula formation, gastrulation, and 2-armed and 4-armed pluteus formation have been analyzed and a number of abnormalities were calculated. Number of embryogenesis anomalies in sand dollar larvae exposed to sea water from different stations in Peter the Great Bay corresponds to pollution level at each area. The Sea of Okhotsk is the main fishing area for Russia. Anthropogenic impact on the marine ecosystem is caused by fishing and transport vessels mainly. But two shelf areas are considered as "hot spots" due to oil and gas drilling. Offshore oil exploitation on the northeastern Sakhalin Island has been started and at present time oil is being drill on oil-extracting platforms continuously. Significant reserves of hydrocarbons are prospected on western Kamchatka shelf, and exploitation drilling in this area was intensified in 2014. A higher number of abnormalities at gastrula and pluteus stages (19-36%) were detected for the stations around oil platforms near Sakhalin Island. On the western Kamchatka shelf number of abnormalities was 7-21%. Such anomalies as exogastrula, incomplete development of pairs of arms were not observed at all; only the delay of development was registered. Eggs, embryos, and larvae of sea urchins are the suitable bioindicators of early disturbances caused by marine pollution in impact ecosystems.

High cell density suppresses BMP4-induced differentiation of human pluripotent stem cells to produce macroscopic spatial patterning in a unidirectional perfusion culture chamber.

PubMed

Tashiro, Shota; Le, Minh Nguyen Tuyet; Kusama, Yuta; Nakatani, Eri; Suga, Mika; Furue, Miho K; Satoh, Taku; Sugiura, Shinji; Kanamori, Toshiyuki; Ohnuma, Kiyoshi

2018-04-19

Spatial pattern formation is a critical step in embryogenesis. Bone morphogenetic protein 4 (BMP4) and its inhibitors are major factors for the formation of spatial patterns during embryogenesis. However, spatial patterning of the human embryo is unclear because of ethical issues and isotropic culture environments resulting from conventional culture dishes. Here, we utilized human pluripotent stem cells (hiPSCs) and a simple anisotropic (unidirectional perfusion) culture chamber, which creates unidirectional conditions, to measure the cell community effect. The influence of cell density on BMP4-induced differentiation was explored during static culture using a conventional culture dish. Immunostaining of the early differentiation marker SSEA-1 and the mesendoderm marker BRACHYURY revealed that high cell density suppressed differentiation, with small clusters of differentiated and undifferentiated cells formed. Addition of five-fold higher concentration of BMP4 showed similar results, suggesting that suppression was not caused by depletion of BMP4 but rather by high cell density. Quantitative RT-PCR array analysis showed that BMP4 induced multi-lineage differentiation, which was also suppressed under high-density conditions. We fabricated an elongated perfusion culture chamber, in which proteins were transported unidirectionally, and hiPSCs were cultured with BMP4. At low density, the expression was the same throughout the chamber. However, at high density, SSEA-1 and BRACHYURY were expressed only in upstream cells, suggesting that some autocrine/paracrine factors inhibited the action of BMP4 in downstream cells to form the spatial pattern. Human iPSCs cultured in a perfusion culture chamber might be useful for studying in vitro macroscopic pattern formation in human embryogenesis. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Early- and Late-Onset Depression in Late Life: A Prospective Study on Clinical and Structural Brain Characteristics and Response to Electroconvulsive Therapy.

PubMed

Dols, Annemiek; Bouckaert, Filip; Sienaert, Pascal; Rhebergen, Didi; Vansteelandt, Kristof; Ten Kate, Mara; de Winter, Francois-Laurent; Comijs, Hannie C; Emsell, Louise; Oudega, Mardien L; van Exel, Eric; Schouws, Sigfried; Obbels, Jasmien; Wattjes, Mike; Barkhof, Frederik; Eikelenboom, Piet; Vandenbulcke, Mathieu; Stek, Max L

2017-02-01

The clinical profile of late-life depression (LLD) is frequently associated with cognitive impairment, aging-related brain changes, and somatic comorbidity. This two-site naturalistic longitudinal study aimed to explore differences in clinical and brain characteristics and response to electroconvulsive therapy (ECT) in early- (EOD) versus late-onset (LOD) late-life depression (respectively onset <55 and ≥55 years). Between January 2011 and December 2013, 110 patients aged 55 years and older with ECT-treated unipolar depression were included in The Mood Disorders in Elderly treated with ECT study. Clinical profile and somatic health were assessed. Magnetic resonance imaging (MRI) scans were performed before the first ECT and visually rated. Response rate was 78.2% and similar between the two sites but significantly higher in LOD compared with EOD (86.9 versus 67.3%). Clinical, somatic, and brain characteristics were not different between EOD and LOD. Response to ECT was associated with late age at onset and presence of psychotic symptoms and not with structural MRI characteristics. In EOD only, the odds for a higher response were associated with a shorter index episode. The clinical profile, somatic comorbidities, and brain characteristics in LLD were similar in EOD and LOD. Nevertheless, patients with LOD showed a superior response to ECT compared with patients with EOD. Our results indicate that ECT is very effective in LLD, even in vascular burdened patients. Copyright © 2017 American Association for Geriatric Psychiatry. Published by Elsevier Inc. All rights reserved.

A comparison of skeletal maturity and growth.

PubMed

Molinari, Luciano; Gasser, Theo; Largo, Remo

2013-07-01

Somatic and bone development have each been studied in detail, but rarely in conjunction. The aim of this study was to determine what somatic and bone development have in common and how they differ. A second aim was to check for a pubertal spurt in bone age (BA) and to quantify it in a similar way as has been done for height. The Preece-Baines model is used to fit longitudinal data for BA. The data analysed are from the 1st Zurich Longitudinal Growth Study comprising 120 boys and 112 girls with almost complete data from birth to adulthood. Variability of somatic milestones was reduced in terms of BA and there was an aftergrowth after reaching adult RUS score 1000. A strong increase in the RUS score was seen at a late stage of the pubertal spurt (PS). Somatic milestones correlated with the RUS score attained at these ages and more so at an early stage of the PS. A PS for BA was clearly identified with a location at 14.2 years for boys and 12.2 years for girls. Age of peak bone development correlated highly with age of peak velocity of somatic variables. BA can be successfully modelled as a semi-quantitative entity. Bone development shows marked associations with somatic development, despite the fact that the latter reflects changes in size, while the former is essentially a maturity index and reflects changes in biochemical composition of tissues.

Variation of mutational burden in healthy human tissues suggests non-random strand segregation and allows measuring somatic mutation rates.

PubMed

Werner, Benjamin; Sottoriva, Andrea

2018-06-01

The immortal strand hypothesis poses that stem cells could produce differentiated progeny while conserving the original template strand, thus avoiding accumulating somatic mutations. However, quantitating the extent of non-random DNA strand segregation in human stem cells remains difficult in vivo. Here we show that the change of the mean and variance of the mutational burden with age in healthy human tissues allows estimating strand segregation probabilities and somatic mutation rates. We analysed deep sequencing data from healthy human colon, small intestine, liver, skin and brain. We found highly effective non-random DNA strand segregation in all adult tissues (mean strand segregation probability: 0.98, standard error bounds (0.97,0.99)). In contrast, non-random strand segregation efficiency is reduced to 0.87 (0.78,0.88) in neural tissue during early development, suggesting stem cell pool expansions due to symmetric self-renewal. Healthy somatic mutation rates differed across tissue types, ranging from 3.5 × 10-9/bp/division in small intestine to 1.6 × 10-7/bp/division in skin.

The Xenopus Maternal-to-Zygotic Transition from the Perspective of the Germline.

PubMed

Yang, Jing; Aguero, Tristan; King, Mary Lou

2015-01-01

In Xenopus, the germline is specified by the inheritance of germ-plasm components synthesized at the beginning of oogenesis. Only the cells in the early embryo that receive germ plasm, the primordial germ cells (PGCs), are competent to give rise to the gametes. Thus, germ-plasm components continue the totipotent potential exhibited by the oocyte into the developing embryo at a time when most cells are preprogrammed for somatic differentiation as dictated by localized maternal determinants. When zygotic transcription begins at the mid-blastula transition, the maternally set program for somatic differentiation is realized. At this time, genetic control is ceded to the zygotic genome, and developmental potential gradually becomes more restricted within the primary germ layers. PGCs are a notable exception to this paradigm and remain transcriptionally silent until the late gastrula. How the germ-cell lineage retains full potential while somatic cells become fate restricted is a tale of translational repression, selective degradation of somatic maternal determinants, and delayed activation of zygotic transcription. © 2015 Elsevier Inc. All rights reserved.

Biological tooth replacement

PubMed Central

Sartaj, Rachel; Sharpe, Paul

2006-01-01

Teeth develop from a series of reciprocal interactions that take place between epithelium and mesenchyme during development of the mouth that begin early in mammalian embryogenesis. The molecular control of key processes in tooth development such as initiation, morphogenesis and cytodifferentiation are being increasingly better understood, to the point where this information can be used as the basis for approaches to produce biological replacement teeth (BioTeeth). This review outlines the current approaches, ideas and progress towards the production of BioTeeth that could form an alternative method for replacing lost or damaged teeth. PMID:17005022