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

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

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.

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.

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

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

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

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

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.

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...

Ultrastructural changes and the distribution of arabinogalactan proteins during somatic embryogenesis of banana (Musa spp. AAA cv. 'Yueyoukang 1').

PubMed

Pan, Xiao; Yang, Xiao; Lin, Guimei; Zou, Ru; Chen, Houbin; Samaj, Jozef; Xu, Chunxiang

2011-08-01

A better understanding of somatic embryogenesis in banana (Musa spp.) may provide a practical way to improve regeneration of banana plants. In this study, we applied scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to visualize the ultrastructural changes during somatic embryogenesis of banana (Musa AAA cv. 'Yueyoukang 1'). We also used histological and immunohistochemical techniques with 16 monoclonal antibodies to study the spatial distribution and cellular/subcellular localization of different arabinogalactan protein (AGP) components of the cell wall during somatic embryogenesis. Histological study with periodic acid-Schiff staining documented diverse embryogenic stages from embryogenic cells (ECs) to the late embryos. SEM revealed a mesh-like structure on the surface of proembryos which represented an early structural marker of somatic embryogenesis. TEM showed that ECs were rich in juvenile mitochondria, endoplasmic reticulum and Golgi stacks. Cells in proembryos and early globular embryos resembled ECs, but they were more vacuolated, showed more regular nuclei and slightly more developed organelles. Immunocytochemical study revealed that the signal of most AGP epitopes was stronger in starch-rich cells when compared with typical ECs. The main AGP component in the extracellular matrix surface network of banana proembryos was the MAC204 epitope. Later, AGP immunolabelling patterns varied with the developmental stages of the embryos. These results about developmental regulation of AGP epitopes along with developmental changes in the ultrastructure of cells are providing new insights into the somatic embryogenesis of banana. Copyright © Physiologia Plantarum 2011.

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.

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.

Carbohydrate-mediated responses during zygotic and early somatic embryogenesis in the endangered conifer, Araucaria angustifolia

PubMed Central

Elbl, Paula; De Souza, Amanda P.; Jardim, Vinicius; de Oliveira, Leandro F.; Macedo, Amanda F.; dos Santos, André L. W.; Buckeridge, Marcos S.; Floh, Eny I. S.

2017-01-01

Three zygotic developmental stages and two somatic Araucaria angustifolia cell lines with contrasting embryogenic potential were analyzed to identify the carbohydrate-mediated responses associated with embryo formation. Using a comparison between zygotic and somatic embryogenesis systems, the non-structural carbohydrate content, cell wall sugar composition and expression of genes involved in sugar sensing were analyzed, and a network analysis was used to identify coordinated features during embryogenesis. We observed that carbohydrate-mediated responses occur mainly during the early stages of zygotic embryo formation, and that during seed development there are coordinated changes that affect the development of the different structures (embryo and megagametophyte). Furthermore, sucrose and starch accumulation were associated with the responsiveness of the cell lines. This study sheds light on how carbohydrate metabolism is influenced during zygotic and somatic embryogenesis in the endangered conifer species, A. angustifolia. PMID:28678868

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.

Developmental Localization and Methylesterification of Pectin Epitopes during Somatic Embryogenesis of Banana (Musa spp. AAA)

PubMed Central

Xu, Chunxiang; Zhao, Lu; Pan, Xiao; Šamaj, Jozef

2011-01-01

Background The plant cell walls play an important role in somatic embryogenesis and plant development. Pectins are major chemical components of primary cell walls while homogalacturonan (HG) is the most abundant pectin polysaccharide. Developmental regulation of HG methyl-esterification degree is important for cell adhesion, division and expansion, and in general for proper organ and plant development. Methodology/Principal Findings Developmental localization of pectic homogalacturonan (HG) epitopes and the (1→4)-β-D-galactan epitope of rhamnogalacturonan I (RG-I) and degree of pectin methyl-esterification (DM) were studied during somatic embryogenesis of banana (Musa spp. AAA). Histological analysis documented all major developmental stages including embryogenic cells (ECs), pre-globular, globular, pear-shaped and cotyledonary somatic embryos. Histochemical staining of extracellularly secreted pectins with ruthenium red showed the most intense staining at the surface of pre-globular, globular and pear-shaped somatic embryos. Biochemical analysis revealed developmental regulation of galacturonic acid content and DM in diverse embryogenic stages. Immunodots and immunolabeling on tissue sections revealed developmental regulation of highly methyl-esterified HG epitopes recognized by JIM7 and LM20 antibodies during somatic embryogenesis. Cell walls of pre-globular/globular and late-stage embryos contained both low methyl-esterified HG epitopes as well as partially and highly methyl-esterified ones. Extracellular matrix which covered surface of early developing embryos contained pectin epitopes recognized by 2F4, LM18, JIM5, JIM7 and LM5 antibodies. De-esterification of cell wall pectins by NaOH caused a decrease or an elimination of immunolabeling in the case of highly methyl-esterified HG epitopes. However, immunolabeling of some low methyl-esterified epitopes appeared stronger after this base treatment. Conclusions/Significance These data suggest that both low- and highly-methyl-esterified HG epitopes are developmentally regulated in diverse embryogenic stages during somatic embryogenesis. This study provides new information about pectin composition, HG methyl-esterification and developmental localization of pectin epitopes during somatic embryogenesis of banana. PMID:21826225

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.

[Changes in polyamine levels in Citrus sinensis Osb. cv. Valencia callus during somatic embryogenesis].

PubMed

Liu, Hua-Ying; Xiao, Lang-Tao; Lu, Xu-Dong; Hu, Jia-Jin; Wu, Shun; He, Chang-Zheng; Deng, Xiu-Xin

2005-06-01

Somatic embryogenetic capability and changes in polyamine level and their relationship were analyzed using the long-term (8 years) subcultured calli of Citrus sinensis Osb. cv. Valencia as materials. The results showed that endogenous polyamine contents in embryogenic calli were higher than those in non-embryogenic calli, and the embryogenetic capability was positively correlated to the levels of endogenous polyamines. When the calli were transferred to a differentiation medium, the putrescine content rapidly increased and reached a peak, then fell gradually. Applying exogenous putrescine raised the embryogenesis frequency and endogenous putrescine level. It indicated that increase in putrescine content at early stage of differentiation promoted embryogenesis. With the development of somatic embryo, spermidine content reached its the highest level at globular embryo stage, spermine content rose and reached a peak at a later stage of globular embryo development. Furthermore, changes of the putrescine, spermidine and spermine contents during somatic embryogenesis were similar in Valencia calli which had different ploidy levels, but their contents decreased following the increasing of ploidy level. Changes in arginine decarboxylase activity were positively correlated to the polyamine levels, which suggest that the later is a key factor in regulating the polyamine levels during somatic embryogenesis in citrus plants.

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.

Somatic embryogenesis in Carica papaya as affected by auxins and explants, and morphoanatomical-related aspects.

PubMed

Cipriano, Jamile L D; Cruz, Ana Cláudia F; Mancini, Karina C; Schmildt, Edilson R; Lopes, José Carlos; Otoni, Wagner C; Alexandre, Rodrigo S

2018-01-01

The aim of this study was to evaluate somatic embryogenesis in juvenile explants of the THB papaya cultivar. Apical shoots and cotyledonary leaves were inoculated in an induction medium composed of different concentrations of 2,4-D (6, 9, 12, 15 and 18 µM) or 4-CPA (19, 22, 25, 28 and 31 µM). The embryogenic calluses were transferred to a maturation medium for 30 days. Histological analysis were done during the induction and scanning electron microscopy after maturing. For both types of auxin, embryogenesis was achieved at higher frequencies with cotyledonary leaves incubated in induction medium than with apical shoots; except for callogenesis. The early-stage embryos (e.g., globular or heart-shape) predominated. Among the auxins, best results were observed in cotyledonary leaves induced with 4-CPA (25 µM). Histological analyses of the cotyledonary leaf-derived calluses confirmed that the somatic embryos (SEs) formed from parenchyma cells, predominantly differentiated via indirect and multicellular origin and infrequently via synchronized embryogenesis. The secondary embryogenesis was observed during induction and maturation phases in papaya THB cultivar. The combination of ABA (0.5 µM) and AC (15 g L-1) in maturation medium resulted in the highest somatic embryogenesis induction frequency (70 SEs callus-1) and the lowest percentage of early germination (4%).

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.

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.

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

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.

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 ...

[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.

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.

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...

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 ...

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.

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.

Effects of copper and arsenic stress on the development of Norway spruce somatic embryos and their visualization with the environmental scanning electron microscope.

PubMed

Đorđević, Biljana; Neděla, Vilém; Tihlaříková, Eva; Trojan, Václav; Havel, Ladislav

2018-05-18

Somatic embryogenesis is an important biotechnological technique which can be used in studies associated with environmental stress. Four embryogenic cell lines of Norway spruce were grown on media enriched with copper and arsenic in concentration ranges 50-500 μM and 10-50 μM, respectively. The effects were observed during subsequent stages of somatic embryogenesis, the characteristics evaluated being proliferation potential, average number of somatic embryos obtained per g/fresh weight, morphology of developed somatic embryos, metal uptake, and microanalysis of macro- and micronutrients uptake. Copper and arsenic at higher concentrations significantly reduced the growth of early somatic embryos. In almost all treatments, the cell line V-1-3 showed the best performance compared with the other lines tested. Environmental scanning electron microscopy was used to visualize and identify morphological abnormalities in the development of somatic embryos. Abnormalities observed were classified into several categories: meristemless somatic embryos, somatic embryos with disrupted meristem, reduced number of cotyledons, single cotyledon and fused cotyledons. With the application of a low temperature method for the environmental scanning electron microscope, samples were stabilized and whole meristems could be investigated in their native state. As far as we are aware, this is the first report of the effect of copper and arsenic during the process of somatic embryogenesis and the first to evaluate the content of macro and micronutrients uptake in Norway spruce. Copyright © 2018 Elsevier B.V. All rights reserved.

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

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.

High stability of nuclear microsatellite loci during the early stages of somatic embryogenesis in Norway spruce.

PubMed

Helmersson, Andreas; von Arnold, Sara; Burg, Kornel; Bozhkov, Peter V

2004-10-01

Somatic embryos of Norway spruce (Picea abies (L.) Karst.) differentiate from proembryogenic masses (PEMs), which are subject to autodestruction through programmed cell death. In PEMs, somatic embryo formation and activation of programmed cell death are interrelated processes. We sought to determine if activation of programmed cell death in PEMs is caused by genetic aberrations during somatic embryogenesis. Based on the finding that withdrawal of auxin and cytokinin induces programmed cell death in PEMs, 1-week-old cell suspensions were cultured in medium either with or without auxin and cytokinin and then transferred to maturation medium containing abscisic acid. We analyzed the stability of three nuclear simple sequence repeat (SSR) microsatellite markers at successive stages of somatic embryogenesis in two cell lines. There were no mutations at the SSR loci at any of the successive developmental stages from PEMs to cotyledonary embryos, irrespective of whether or not the proliferation medium in which cell suspensions had been cultured contained auxin or cytokinin. The morphologies of plants regenerated from the cultures were similar, although withdrawal of auxin and cytokinin significantly stimulated the yield of both embryos and plants. We conclude, therefore, that the high genetic stability of somatic embryos in Norway spruce is unaffected by the induction of programmed cell death caused by withdrawal of auxin and cytokinin.

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.

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.

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.

Ectopic expression of the Coffea canephora SERK1 homolog-induced differential transcription of genes involved in auxin metabolism and in the developmental control of embryogenesis.

PubMed

Pérez-Pascual, Daniel; Jiménez-Guillen, Doribet; Villanueva-Alonzo, Hernán; Souza-Perera, Ramón; Godoy-Hernández, Gregorio; Zúñiga-Aguilar, José Juan

2018-04-01

Somatic embryogenesis receptor-like kinase 1 (SERK1) is a membrane receptor that might serve as common co-regulator of plant cell differentiation processes by forming heterodimers with specific receptor-like kinases. The Coffea canephora SERK1 homolog (CcSERK1) was cloned in this work, and its early function in the transcription of embryogenesis master genes and of genes encoding proteins involved in auxin metabolism was investigated by externally manipulating its expression in embryogenic leaf explants, before the appearance of embryogenic structures. Overexpression of CcSERK1 early during embryogenesis caused an increase in the number of somatic embryos when the 55-day process was completed. Suppression of CcSERK1 expression by RNA interference almost abolished somatic embryogenesis. Real time-PCR experiments revealed that the transcription of the CcAGL15, CcWUS, CcBBM, CcPKL, CcYUC1, CcPIN1 and CcPIN4 homologs was modified in direct proportion to the expression of CcSERK1 and that only CcLEC1 was inversely affected by the expression levels of CcSERK1. The expression of the CcYUC4 homolog was induced to more than 80-fold under CcSERK1 overexpression conditions, but it was also induced when CcSERK1 expression was silenced. The level of CcTIR1 was not affected by CcSERK1 overexpression but was almost abolished during CcSERK1 silencing. These results suggest that CcSERK1 co-regulates the induction of somatic embryogenesis in Coffea canephora by early activation of YUC-dependent auxin biosynthesis, auxin transport mediated by PIN1 and PIN4, and probably auxin perception by the TIR1 receptor, leading to the induction of early-stage homeotic genes (CcAGL15, CcWUS, CcPKL and CcBBM) and repression of late-stage homeotic genes (CcLec1). © 2018 Scandinavian Plant Physiology Society.

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.

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.

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

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.

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.

The role of chromatin modifications in somatic embryogenesis in plants

PubMed Central

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

2015-01-01

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

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.

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.

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.

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.

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

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.

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.

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

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.

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.

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

Setting the Clock for Fail-Safe Early Embryogenesis.

PubMed

Fickentscher, Rolf; Struntz, Philipp; Weiss, Matthias

2016-10-28

The embryogenesis of the small nematode Caenorhabditis elegans is a remarkably robust self-organization phenomenon. Cell migration trajectories in the early embryo, for example, are well explained by mechanical cues that push cells into positions where they experience the least repulsive forces. Yet, how this mechanically guided progress in development is properly timed has remained elusive so far. Here, we show that cell volumes and division times are strongly anticorrelated during the early embryogenesis of C. elegans with significant differences between somatic cells and precursors of the germline. Our experimental findings are explained by a simple model that in conjunction with mechanical guidance can account for the fail-safe early embryogenesis of C. elegans.

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.

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.

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...

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.

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.

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.

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.

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

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

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.

Somatic mutations reveal asymmetric cellular dynamics in the early human embryo

DOE PAGES

Ju, Young Seok; Martincorena, Inigo; Gerstung, Moritz; ...

2017-03-22

Somatic cells acquire mutations throughout the course of an individual’s life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and theirmore » contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. As a result, this study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.« less

Somatic mutations reveal asymmetric cellular dynamics in the early human embryo

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

Ju, Young Seok; Martincorena, Inigo; Gerstung, Moritz

Somatic cells acquire mutations throughout the course of an individual’s life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and theirmore » contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. As a result, this study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.« less

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.

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

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

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.

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.

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.

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.

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.

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.

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...

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.

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

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.

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.

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.

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...

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

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,...

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.

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

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...

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.

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.

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

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

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/.

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.

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

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

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.

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

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.

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.

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

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.

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.

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.

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...

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.

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.

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.

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.

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.

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.

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.

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.

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

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

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.

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.

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

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.

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

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

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

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.

Zinc tolerance and accumulation in stable cell suspension cultures and in vitro regenerated plants of the emerging model plant Arabidopsis halleri (Brassicaceae).

PubMed

Vera-Estrella, Rosario; Miranda-Vergara, Maria Cristina; Barkla, Bronwyn J

2009-03-01

Arabidopsis halleri is increasingly employed as a model plant for studying heavy metal hyperaccumulation. With the aim of providing valuable tools for studies on cellular physiology and molecular biology of metal tolerance and transport, this study reports the development of successful and highly efficient methods for the in vitro regeneration of A. halleri plants and production of stable cell suspension lines. Plants were regenerated from leaf explants of A. halleri via a three-step procedure: callus induction, somatic embryogenesis and shoot development. Efficiency of callus proliferation and regeneration depended on the initial callus induction media and was optimal in the presence of 1 mg L(-1) 2,4-dichlorophenoxyacetic acid, and 0.05 mg L(-1) benzylaminopurine. Subsequent shoot and root regeneration from callus initiated under these conditions reached levels of 100% efficiency. High friability of the callus supported the development of cell suspension cultures with minimal cellular aggregates. Characterization of regenerated plants and cell cultures determined that they maintained not only the zinc tolerance and requirement of the whole plant but also the ability to accumulate zinc; with plants accumulating up to 50.0 micromoles zinc g(-1) FW, and cell suspension cultures 30.9 micromoles zinc g(-1) DW. Together this work will provide the experimental basis for furthering our knowledge of A. halleri as a model heavy metal hyperaccumulating plant.

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

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.

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.

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.

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

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.

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

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

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

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

Somatic embryogenesis and massive shoot regeneration from immature embryo explants of tef.

PubMed

Gugsa, Likyelesh; Kumlehn, Jochen

2011-01-01

Tef (Eragrostis tef) provides a major source of human nutrition in the Horn of Africa, but biotechnology has had little impact on its improvement to date. Here, we report the elaboration of an in vitro regeneration protocol, based on the use of immature zygotic embryos as explant. Explant size was an important determinant of in vitro regeneration efficiency, as was the formulation of the culture medium. Optimal results were obtained by culturing 0.2-0.35 mm embryo explants on a medium containing KBP minerals, 9.2-13.8 μM 2,4-dichlorophenoxyacetic acid, 6 mM glutamine, and 0.5% Phytagel. Although this protocol was effective for both the improved cultivar "DZ-01-196" and the landrace "Fesho", the former produced consistently more embryogenic tissue and a higher number of regenerants. An average of more than 2,800 shoots could be obtained from each "DZ-01-196" explant after 12 weeks of in vitro culture. These shoots readily formed roots, and plantlets transferred to soil were able to develop into morphologically normal, fertile plants. This regeneration and multiplication system should allow for the application of a range of biotechnological methods to tef.

Functional analysis of the GmESR1 gene associated with soybean regeneration

PubMed Central

Chen, Qingshan; Liu, Ming; Xin, Dawei; Qi, Zhaoming; Li, Sinan; Ma, Yanlong; Wang, Lingshuang; Jin, Yangmei; Li, Wenbin; Wu, Xiaoxia; Su, An-yu

2017-01-01

Plant regeneration can occur via in vitro tissue culture through somatic embryogenesis or de novo shoot organogenesis. Transformation of soybean (Glycine max) is difficult, hence optimization of the transformation system for soybean regeneration is required. This study investigated ENHANCER OF SHOOT REGENERATION 1 (GmESR1), a soybean transcription factor that targets regeneration-associated genes. Sequence analysis showed that GmESR1 contained a conserved 57 amino acid APETALA 2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) DNA-binding domain. The relative expression level of GmESR1 was highest in young embryos, flowers and stems in the soybean cultivar ‘Dongnong 50’. To examine the function of GmESR1, transgenic Arabidopsis (Arabidopsis thaliana) and soybean plants overexpressing GmESR1 were generated. In Arabidopsis, overexpression of GmESR1 resulted in accelerated seed germination, and seedling shoot and root elongation. In soybean overexpression of GmESR1 also led to faster seed germination, and shoot and root elongation. GmESR1 specifically bound to the GCC-box. The results provide a foundation for the establishment of an efficient and stable transformation system for soybean. PMID:28403182

Biotechnological applications in in vitro plant regeneration studies of broccoli (Brassica oleracea L. var. italica), an important vegetable crop.

PubMed

Kumar, Pankaj; Srivastava, Dinesh Kumar

2016-04-01

Biotechnology holds promise for genetic improvement of important vegetable crops. Broccoli (Brassica oleracea L. var. italica) is an important vegetable crop of the family Brassicaceae. However, various biotic and abiotic stresses cause enormous crop yield losses during commercial cultivation of broccoli. Establishment of a reliable, reproducible and efficient in vitro plant regeneration system with cell and tissue culture is a vital prerequisite for biotechnological application of crop improvement programme. An in vitro plant regeneration technique refers to culturing, cell division, cell multiplication, de-differentiation and differentiation of cells, protoplasts, tissues and organs on defined liquid/solid medium under aseptic and controlled environment. Recent progress in the field of plant tissue culture has made this area one of the most dynamic and promising in experimental biology. There are many published reports on in vitro plant regeneration studies in broccoli including direct organogenesis, indirect organogenesis and somatic embryogenesis. This review summarizes those plant regeneration studies in broccoli that could be helpful in drawing the attention of the researchers and scientists to work on it to produce healthy, biotic and abiotic stress resistant plant material and to carry out genetic transformation studies for the production of transgenic plants.

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

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.

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

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

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.

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

Distribution of potato spindle tuber viroid in reproductive organs of petunia during its developmental stages.

PubMed

Matsushita, Yosuke; Tsuda, Shinya

2014-09-01

Embryo infection is important for efficient seed transmission of viroids. To identify the major pattern of seed transmission of viroids, we used in situ hybridization to histochemically analyze the distribution of Potato spindle tuber viroid (PSTVd) in each developmental stage of petunia (flowering to mature seed stages). In floral organs, PSTVd was present in the reproductive tissues of infected female × infected male and infected female × healthy male but not of healthy female × infected male before embryogenesis. After pollination, PSTVd was detected in the developed embryo and endosperm in all three crosses. These findings indicate that PSTVd is indirectly delivered to the embryo through ovule or pollen during the development of reproductive tissues before embryogenesis but not directly through maternal tissues as cell-to-cell movement during embryogenesis.

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

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.

Privileged Communication Embryonic Development Following Somatic Cell Nuclear Transfer Impeded by Persisting Histone Methylation

PubMed Central

Matoba, Shogo; Liu, Yuting; Lu, Falong; Iwabuchi, Kumiko A.; Shen, Li; Inoue, Azusa; Zhang, Yi

2014-01-01

SUMMARY Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major epigenetic barrier for efficient reprogramming by SCNT. Comparative transcriptome analysis identified reprogramming resistant regions (RRRs) that are expressed normally at 2-cell mouse embryos generated by IVF but not SCNT. RRRs are enriched for H3K9me3 in donor somatic cells, and its removal by ectopic expression of the H3K9me3 demethylase Kdm4d not only reactivates the majority of RRRs, but also greatly improves SCNT efficiency. Furthermore, use of donor somatic nuclei depleted of H3K9 methyltransferases markedly improves SCNT efficiency. Our study thus identifies H3K9me3 as a critical epigenetic barrier in SCNT-mediated reprogramming and provides a promising approach for improving mammalian cloning efficiency. PMID:25417163

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.

Somatic Embryogenesis and Massive Shoot Regeneration from Immature Embryo Explants of Tef

PubMed Central

Gugsa, Likyelesh; Kumlehn, Jochen

2011-01-01

Tef (Eragrostis tef) provides a major source of human nutrition in the Horn of Africa, but biotechnology has had little impact on its improvement to date. Here, we report the elaboration of an in vitro regeneration protocol, based on the use of immature zygotic embryos as explant. Explant size was an important determinant of in vitro regeneration efficiency, as was the formulation of the culture medium. Optimal results were obtained by culturing 0.2–0.35 mm embryo explants on a medium containing KBP minerals, 9.2–13.8 μM 2,4-dichlorophenoxyacetic acid, 6 mM glutamine, and 0.5% Phytagel. Although this protocol was effective for both the improved cultivar “DZ-01-196” and the landrace “Fesho”, the former produced consistently more embryogenic tissue and a higher number of regenerants. An average of more than 2,800 shoots could be obtained from each “DZ-01-196” explant after 12 weeks of in vitro culture. These shoots readily formed roots, and plantlets transferred to soil were able to develop into morphologically normal, fertile plants. This regeneration and multiplication system should allow for the application of a range of biotechnological methods to tef. PMID:22028975

Organogenic nodule formation in hop: a tool to study morphogenesis in plants with biotechnological and medicinal applications.

PubMed

Fortes, Ana M; Santos, Filipa; Pais, Maria S

2010-01-01

The usage of Humulus lupulus for brewing increased the demand for high-quality plant material. Simultaneously, hop has been used in traditional medicine and recently recognized with anticancer and anti-infective properties. Tissue culture techniques have been reported for a wide range of species, and open the prospect for propagation of disease-free, genetically uniform and massive amounts of plants in vitro. Moreover, the development of large-scale culture methods using bioreactors enables the industrial production of secondary metabolites. Reliable and efficient tissue culture protocol for shoot regeneration through organogenic nodule formation was established for hop. The present review describes the histological, and biochemical changes occurring during this morphogenic process, together with an analysis of transcriptional and metabolic profiles. We also discuss the existence of common molecular factors among three different morphogenic processes: organogenic nodules and somatic embryogenesis, which strictly speaking depend exclusively on intrinsic developmental reprogramming, and legume nitrogen-fixing root nodules, which arises in response to symbiosis. The review of the key factors that participate in hop nodule organogenesis and the comparison with other morphogenic processes may have merit as a study presenting recent advances in complex molecular networks occurring during morphogenesis and together, these provide a rich framework for biotechnology applications.

Factors affecting the development of somatic cell nuclear transfer embryos in Cattle.

PubMed

Akagi, Satoshi; Matsukawa, Kazutsugu; Takahashi, Seiya

2014-01-01

Nuclear transfer is a complex multistep procedure that includes oocyte maturation, cell cycle synchronization of donor cells, enucleation, cell fusion, oocyte activation and embryo culture. Therefore, many factors are believed to contribute to the success of embryo development following nuclear transfer. Numerous attempts to improve cloning efficiency have been conducted since the birth of the first sheep by somatic cell nuclear transfer. However, the efficiency of somatic cell cloning has remained low, and applications have been limited. In this review, we discuss some of the factors that affect the developmental ability of somatic cell nuclear transfer embryos in cattle.

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

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.

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.

Agrobacterium rhizogenes-mediated transformation of Superroot-derived Lotus corniculatus plants: a valuable tool for functional genomics.

PubMed

Jian, Bo; Hou, Wensheng; Wu, Cunxiang; Liu, Bin; Liu, Wei; Song, Shikui; Bi, Yurong; Han, Tianfu

2009-06-25

Transgenic approaches provide a powerful tool for gene function investigations in plants. However, some legumes are still recalcitrant to current transformation technologies, limiting the extent to which functional genomic studies can be performed on. Superroot of Lotus corniculatus is a continuous root cloning system allowing direct somatic embryogenesis and mass regeneration of plants. Recently, a technique to obtain transgenic L. corniculatus plants from Superroot-derived leaves through A. tumefaciens-mediated transformation was described. However, transformation efficiency was low and it took about six months from gene transfer to PCR identification. In the present study, we developed an A. rhizogenes-mediated transformation of Superroot-derived L. corniculatus for gene function investigation, combining the efficient A. rhizogenes-mediated transformation and the rapid regeneration system of Superroot. The transformation system using A. rhizogenes K599 harbouring pGFPGUSPlus was improved by validating some parameters which may influence the transformation frequency. Using stem sections with one node as explants, a 2-day pre-culture of explants, infection with K599 at OD(600) = 0.6, and co-cultivation on medium (pH 5.4) at 22 degrees C for 2 days enhanced the transformation frequency significantly. As proof of concept, Superroot-derived L. corniculatus was transformed with a gene from wheat encoding an Na+/H+ antiporter (TaNHX2) using the described system. Transgenic Superroot plants were obtained and had increased salt tolerance, as expected from the expression of TaNHX2. A rapid and efficient tool for gene function investigation in L. corniculatus was developed, combining the simplicity and high efficiency of the Superroot regeneration system and the availability of A. rhizogenes-mediated transformation. This system was improved by validating some parameters influencing the transformation frequency, which could reach 92% based on GUS detection. The combination of the highly efficient transformation and the regeneration system of Superroot provides a valuable tool for functional genomics studies in L. corniculatus.

Optimisation of tomato Micro-tom regeneration and selection on glufosinate/Basta and dependency of gene silencing on transgene copy number.

PubMed

Khuong, Thi Thu Huong; Crété, Patrice; Robaglia, Christophe; Caffarri, Stefano

2013-09-01

An efficient protocol of transformation and selection of transgenic lines of Micro-tom, a widespread model cultivar for tomato, is reported. RNA interference silencing efficiency and stability have been investigated and correlated with the number of insertions. Given its small size and ease of cultivation, the tomato (Solanum lycopersicon) cultivar Micro-tom is of widespread use as a model tomato plant. To create and screen transgenic plants, different selectable markers are commonly used. The bar marker carrying the resistance to the herbicide glufosinate/Basta, has many advantages, but it has been little utilised and with low efficiency for identification of tomato transgenic plants. Here we describe a procedure for accurate selection of transgenic Micro-tom both in vitro and in soil. Immunoblot, Southern blot and phenotypic analyses showed that 100 % of herbicide-resistant plants were transgenic. In addition, regeneration improvement has been obtained by using 2 mg/l Gibberellic acid in the shoot elongation medium; rooting optimisation on medium containing 1 mg/l IAA allowed up to 97 % of shoots developing strong and very healthy roots after only 10 days. Stable transformation frequency by infection of leaf explants with Agrobacterium reached 12 %. Shoots have been induced by combination of 1 mg/l zeatin-trans and 0.1 mg/l IAA. Somatic embryogenesis of cotyledon on medium containing 1 mg/l zeatin + 2 mg/l IAA is described in Micro-tom. The photosynthetic psbS gene has been used as reporter gene for RNA silencing studies. The efficiency of gene silencing has been found equivalent using three different target gene fragments of 519, 398 and 328 bp. Interestingly, silencing efficiency decreased from T0 to the T3 generation in plants containing multiple copies of the inserted T-DNA, while it was stable in plants containing a single insertion.

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

PubMed Central

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

2015-01-01

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

Factors Affecting the Development of Somatic Cell Nuclear Transfer Embryos in Cattle

PubMed Central

AKAGI, Satoshi; MATSUKAWA, Kazutsugu; TAKAHASHI, Seiya

2014-01-01

Nuclear transfer is a complex multistep procedure that includes oocyte maturation, cell cycle synchronization of donor cells, enucleation, cell fusion, oocyte activation and embryo culture. Therefore, many factors are believed to contribute to the success of embryo development following nuclear transfer. Numerous attempts to improve cloning efficiency have been conducted since the birth of the first sheep by somatic cell nuclear transfer. However, the efficiency of somatic cell cloning has remained low, and applications have been limited. In this review, we discuss some of the factors that affect the developmental ability of somatic cell nuclear transfer embryos in cattle. PMID:25341701

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.

Differentiated cells are more efficient than adult stem cells for cloning by somatic cell nuclear transfer.

PubMed

Sung, Li-Ying; Gao, Shaorong; Shen, Hongmei; Yu, Hui; Song, Yifang; Smith, Sadie L; Chang, Ching-Chien; Inoue, Kimiko; Kuo, Lynn; Lian, Jin; Li, Ao; Tian, X Cindy; Tuck, David P; Weissman, Sherman M; Yang, Xiangzhong; Cheng, Tao

2006-11-01

Since the creation of Dolly via somatic cell nuclear transfer (SCNT), more than a dozen species of mammals have been cloned using this technology. One hypothesis for the limited success of cloning via SCNT (1%-5%) is that the clones are likely to be derived from adult stem cells. Support for this hypothesis comes from the findings that the reproductive cloning efficiency for embryonic stem cells is five to ten times higher than that for somatic cells as donors and that cloned pups cannot be produced directly from cloned embryos derived from differentiated B and T cells or neuronal cells. The question remains as to whether SCNT-derived animal clones can be derived from truly differentiated somatic cells. We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells, progenitor cells and granulocytes. We found that cloning efficiency increases over the differentiation hierarchy, and terminally differentiated postmitotic granulocytes yield cloned pups with the greatest cloning efficiency.

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

PubMed

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

2016-04-01

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

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.

Excessive Cellular Proliferation Negatively Impacts Reprogramming Efficiency of Human Fibroblasts

PubMed Central

Gupta, Manoj K.; Teo, Adrian Kee Keong; Rao, Tata Nageswara; Bhatt, Shweta; Kleinridders, Andre; Shirakawa, Jun; Takatani, Tomozumi; Hu, Jiang; De Jesus, Dario F.; Windmueller, Rebecca; Wagers, Amy J.

2015-01-01

The impact of somatic cell proliferation rate on induction of pluripotent stem cells remains controversial. Herein, we report that rapid proliferation of human somatic fibroblasts is detrimental to reprogramming efficiency when reprogrammed using a lentiviral vector expressing OCT4, SOX2, KLF4, and cMYC in insulin-rich defined medium. Human fibroblasts grown in this medium showed higher proliferation, enhanced expression of insulin signaling and cell cycle genes, and a switch from glycolytic to oxidative phosphorylation metabolism, but they displayed poor reprogramming efficiency compared with cells grown in normal medium. Thus, in contrast to previous studies, our work reveals an inverse correlation between the proliferation rate of somatic cells and reprogramming efficiency, and also suggests that upregulation of proteins in the growth factor signaling pathway limits the ability to induce pluripotency in human somatic fibroblasts. Significance The efficiency with which human cells can be reprogrammed is of interest to stem cell biology. In this study, human fibroblasts cultured in media containing different concentrations of growth factors such as insulin and insulin-like growth factor-1 exhibited variable abilities to proliferate, with consequences on pluripotency. This occurred in part because of changes in the expression of proteins involved in the growth factor signaling pathway, glycolysis, and oxidative phosphorylation. These findings have implications for efficient reprogramming of human cells. PMID:26253715

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.

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.

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.

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.

In Vitro Fertilization with Isolated, Single Gametes Results in Zygotic Embryogenesis and Fertile Maize Plants.

PubMed Central

Kranz, E; Lorz, H

1993-01-01

We demonstrate here the possibility of regenerating phenotypically normal, fertile maize plants via in vitro fertilization of isolated, single sperm and egg cells mediated by electrofusion. The technique leads to the highly efficient formation of polar zygotes, globular structures, proembryos, and transition-phase embryos and to the formation of plants from individually cultured fusion products. Regeneration of plants occurs via embryogenesis and occasionally by polyembryony and organogenesis. Flowering plants can be obtained within 100 days of gamete fusion. Regenerated plants were studied by karyological and morphological analyses, and the segregation of kernel color was determined. The hybrid nature of the plants was confirmed. PMID:12271084

The cell agglutination agent, phytohemagglutinin-L, improves the efficiency of somatic nuclear transfer cloning in cattle (Bos taurus).

PubMed

Du, Fuliang; Shen, Perng-Chih; Xu, Jie; Sung, Li-Ying; Jeong, B-Seon; Lucky Nedambale, Tshimangadzo; Riesen, John; Cindy Tian, X; Cheng, Winston T K; Lee, Shan-Nan; Yang, Xiangzhong

2006-02-01

One of the several factors that contribute to the low efficiency of mammalian somatic cloning is poor fusion between the small somatic donor cell and the large recipient oocyte. This study was designed to test phytohemagglutinin (PHA) agglutination activity on fusion rate, and subsequent developmental potential of cloned bovine embryos. The toxicity of PHA was established by examining its effects on the development of parthenogenetic bovine oocytes treated with different doses (Experiment 1), and for different durations (Experiment 2). The effective dose and duration of PHA treatment (150 microg/mL, 20 min incubation) was selected and used to compare membrane fusion efficiency and embryo development following somatic cell nuclear transfer (Experiment 3). Cloning with somatic donor fibroblasts versus cumulus cells was also compared, both with and without PHA treatment (150 microg/mL, 20 min). Fusion rate of nuclear donor fibroblasts, after phytohemagglutinin treatment, was increased from 33 to 61% (P < 0.05), and from 59 to 88% (P < 0.05) with cumulus cell nuclear donors. The nuclear transfer (NT) efficiency per oocyte used was improved following PHA treatment, for both fibroblast (13% versus 22%) as well as cumulus cells (17% versus 34%; P < 0.05). The cloned embryos, both with and without PHA treatment, were subjected to vitrification and embryo transfer testing, and resulted in similar survival (approximately 90% hatching) and pregnancy rates (17-25%). Three calves were born following vitrification and embryo transfer of these embryos; two from the PHA-treated group, and one from non-PHA control group. We concluded that PHA treatment significantly improved the fusion efficiency of somatic NT in cattle, and therefore, increased the development of cloned blastocysts. Furthermore, within a determined range of dose and duration, PHA had no detrimental effect on embryo survival post-vitrification, nor on pregnancy or calving rates following embryo transfer.

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.

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.

[Predictors of efficiency of autogenous melodeclamation training in patients with chronic somatic pathology].

PubMed

Trdatian, N A

2009-01-01

This controlled study involving 99 patients with chronic somatic diseases (CSD) had the objective to identify psychological predictors of the efficiency of a new method of psychotherapy, namely autogenous melodeclamation training (AMDT). Dynamics of the psychological status of the patients in the course of therapy was assessed using SMOL test, Spilberger STAI test, overall health-physical activity-mood test, and Beck depression inventory. It was shown that moderately compromised psychological adaptation and minor depressive disorders were the most reliable predictors of marked improvement of the patients' psychological status under effect of autogenous melodeclamation training included in the combined rehabilitative therapy of chronic somatic diseases.

Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells

PubMed Central

Chung, H.J.; Hassan, M.M.; Park, J.O.; Kim, H.J.; Hong, S.T.

2015-01-01

Recent advances have raised hope that transplantation of adherent somatic cells could provide dramatic new therapies for various diseases. However, current methods for transplanting adherent somatic cells are not efficient enough for therapeutic applications. Here, we report the development of a novel method to generate quasi-natural cell blocks for high-efficiency transplantation of adherent somatic cells. The blocks were created by providing a unique environment in which cultured cells generated their own extracellular matrix. Initially, stromal cells isolated from mice were expanded in vitro in liquid cell culture medium followed by transferring the cells into a hydrogel shell. After incubation for 1 day with mechanical agitation, the encapsulated cell mass was perforated with a thin needle and then incubated for an additional 6 days to form a quasi-natural cell block. Allograft transplantation of the cell block into C57BL/6 mice resulted in perfect adaptation of the allograft and complete integration into the tissue of the recipient. This method could be widely applied for repairing damaged cells or tissues, stem cell transplantation, ex vivo gene therapy, or plastic surgery. PMID:25742639

Protocol for efficient regulation of in vitro morphogenesis in einkorn (Triticum monococcum L.), a recalcitrant diploid wheat species

PubMed Central

Miroshnichenko, Dmitry; Chaban, Inna; Chernobrovkina, Mariya; Dolgov, Sergey

2017-01-01

Einkorn (Triticum monococcum L.) is A-genome diploid wheat that has a potential to become a useful model for understanding the biology and genomics in Triticeae. Unfortunately, the application of modern technologies such as genetic engineering, RNAi-based gene silencing and genome editing is not available for einkorn as there is no efficient in vitro tissue culture and plant regeneration system. In the present study an efficient and simple protocol for plant regeneration via direct or indirect somatic embryogenesis and organogenesis has been developed. Various auxins used as sole inductors in einkorn displayed low effect for morphogenesis (0–8%) and plant regeneration (1–2 shoots per explant). The addition of Daminozide, the inhibitor of biosynthesis of gibberellins, together with auxin significantly improved the formation of morphogenic structures, especially when Dicamba (51.4%) and Picloram (56.6%) were used for combination; furthermore, the simultaneous addition of cytokinin into induction medium significantly promoted in vitro performance. Among the tested cytokinins, the urea-type substances, such as TDZ and CPPU were more effective than the adenine type ones, BA and Zeatin, for the regulation of morphogenesis; especially, TDZ was more effective than CPPU for shoot formation (11.73 vs. 7.04 per regenerating callus). The highest morphogenic response of 90.2% with the production of more than 10 shoots per initial explant was observed when 3.0 mg/L Dicamba, 50.0 mg/L Daminozide and 0.25 mg/L TDZ were combined together. Along with the identification of appropriate induction medium, the optimal developmental stage for einkorn was found as partially transparent immature embryo in size of around 1.0 mm. Although in the present study the critical balance between plant growth regulators was established for einkorn only, we assume that further the proposed strategy could be successfully applied to other recalcitrant cereal species and genotypes. PMID:28273182

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.

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.

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

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

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.

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

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

Neural network classification of sweet potato embryos

NASA Astrophysics Data System (ADS)

Molto, Enrique; Harrell, Roy C.

1993-05-01

Somatic embryogenesis is a process that allows for the in vitro propagation of thousands of plants in sub-liter size vessels and has been successfully applied to many significant species. The heterogeneity of maturity and quality of embryos produced with this technique requires sorting to obtain a uniform product. An automated harvester is being developed at the University of Florida to sort embryos in vitro at different stages of maturation in a suspension culture. The system utilizes machine vision to characterize embryo morphology and a fluidic based separation device to isolate embryos associated with a pre-defined, targeted morphology. Two different backpropagation neural networks (BNN) were used to classify embryos based on information extracted from the vision system. One network utilized geometric features such as embryo area, length, and symmetry as inputs. The alternative network utilized polar coordinates of an embryo's perimeter with respect to its centroid as inputs. The performances of both techniques were compared with each other and with an embryo classification method based on linear discriminant analysis (LDA). Similar results were obtained with all three techniques. Classification efficiency was improved by reducing the dimension of the feature vector trough a forward stepwise analysis by LDA. In order to enhance the purity of the sample selected as harvestable, a reject to classify option was introduced in the model and analyzed. The best classifier performances (76% overall correct classifications, 75% harvestable objects properly classified, homogeneity improvement ratio 1.5) were obtained using 8 features in a BNN.

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.

EHMT2 directs DNA methylation for efficient gene silencing in mouse embryos

PubMed Central

Auclair, Ghislain; Borgel, Julie; Sanz, Lionel A.; Vallet, Judith; Guibert, Sylvain; Dumas, Michael; Cavelier, Patricia; Girardot, Michael; Forné, Thierry; Feil, Robert; Weber, Michael

2016-01-01

The extent to which histone modifying enzymes contribute to DNA methylation in mammals remains unclear. Previous studies suggested a link between the lysine methyltransferase EHMT2 (also known as G9A and KMT1C) and DNA methylation in the mouse. Here, we used a model of knockout mice to explore the role of EHMT2 in DNA methylation during mouse embryogenesis. The Ehmt2 gene is expressed in epiblast cells but is dispensable for global DNA methylation in embryogenesis. In contrast, EHMT2 regulates DNA methylation at specific sequences that include CpG-rich promoters of germline-specific genes. These loci are bound by EHMT2 in embryonic cells, are marked by H3K9 dimethylation, and have strongly reduced DNA methylation in Ehmt2−/− embryos. EHMT2 also plays a role in the maintenance of germline-derived DNA methylation at one imprinted locus, the Slc38a4 gene. Finally, we show that DNA methylation is instrumental for EHMT2-mediated gene silencing in embryogenesis. Our findings identify EHMT2 as a critical factor that facilitates repressive DNA methylation at specific genomic loci during mammalian development. PMID:26576615

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.

Effect of ovary induction on bread wheat anther culture: ovary genotype and developmental stage, and candidate gene association

PubMed Central

Castillo, Ana M.; Sánchez-Díaz, Rosa A.; Vallés, María P.

2015-01-01

Ovary pre-conditioned medium and ovary co-culture increased the efficiency of green doubled haploid plant production in bread wheat anther culture. The positive effect of this medium led to a 6- and 11-fold increase in the numbers of embryos and green plants, respectively, having a greater effect on a medium-low responding cultivar. Ovary genotype and developmental stage significantly affected microspore embryogenesis. By the use of Caramba ovaries it was possible to reach a 2-fold increase in the number of embryos and green plants, and to decrease the rate of albinism. Mature ovaries from flowers containing microspores at a late binucleate stage raised the number of embryos and green plants by 25–46% as compared to immature ovaries (excised from flowers with microspores at a mid-late uninucleate stage). The highest numbers of embryos and green plants were produced when using mature Caramba ovaries. Ovaries from Galeón, Tigre, and Kilopondio cultivars successfully induced microspore embryogenesis at the same rate as Caramba ovaries. Moreover, Tigre ovaries raised the percentage of spontaneous chromosome doubling up to 71%. Attempts were made to identify molecular mechanisms associated to the inductive effect of the ovaries on microspore embryogenesis. The genes TAA1b, FLA26, and WALI6 associated to wheat microspore embryogenesis, the CGL1 gene involved in glycan biosynthesis or degradation, and the FER gene involved in the ovary signaling process were expressed and/or induced at different rates during ovary culture. The expression pattern of FLA26 and FER could be related to the differences between genotypes and developmental stages in the inductive effect of the ovary. Our results open opportunities for new approaches to increase bread wheat doubled haploid production by anther culture, and to identify the functional components of the ovary inductive effect on microspore embryogenesis. PMID:26150821

Genome editing reveals a role for OCT4 in human embryogenesis.

PubMed

Fogarty, Norah M E; McCarthy, Afshan; Snijders, Kirsten E; Powell, Benjamin E; Kubikova, Nada; Blakeley, Paul; Lea, Rebecca; Elder, Kay; Wamaitha, Sissy E; Kim, Daesik; Maciulyte, Valdone; Kleinjung, Jens; Kim, Jin-Soo; Wells, Dagan; Vallier, Ludovic; Bertero, Alessandro; Turner, James M A; Niakan, Kathy K

2017-10-05

Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.

Simple Monitoring of Gene Targeting Efficiency in Human Somatic Cell Lines Using the PIGA Gene

PubMed Central

Karnan, Sivasundaram; Konishi, Yuko; Ota, Akinobu; Takahashi, Miyuki; Damdindorj, Lkhagvasuren; Hosokawa, Yoshitaka; Konishi, Hiroyuki

2012-01-01

Gene targeting in most of human somatic cell lines has been labor-intensive because of low homologous recombination efficiency. The development of an experimental system that permits a facile evaluation of gene targeting efficiency in human somatic cell lines is the first step towards the improvement of this technology and its application to a broad range of cell lines. In this study, we utilized phosphatidylinositol glycan anchor biosynthesis class A (PIGA), a gene essential for the synthesis of glycosylphosphatidyl inositol (GPI) anchors, as a reporter of gene targeting events in human somatic cell lines. Targeted disruption of PIGA was quantitatively detected with FLAER, a reagent that specifically binds to GPI anchors. Using this PIGA-based reporter system, we successfully detected adeno-associated virus (AAV)-mediated gene targeting events both with and without promoter-trap enrichment of gene-targeted cell population. The PIGA-based reporter system was also capable of reproducing previous findings that an AAV-mediated gene targeting achieves a remarkably higher ratio of homologous versus random integration (H/R ratio) of targeting vectors than a plasmid-mediated gene targeting. The PIGA-based system also detected an approximately 2-fold increase in the H/R ratio achieved by a small negative selection cassette introduced at the end of the AAV-based targeting vector with a promoter-trap system. Thus, our PIGA-based system is useful for monitoring AAV-mediated gene targeting and will assist in improving gene targeting technology in human somatic cell lines. PMID:23056640

Climbing Mount Efficiency--small steps, not giant leaps towards higher cloning success in farm animals.

PubMed

Oback, Björn

2008-07-01

Despite more than a decade of research efforts, farm animal cloning by somatic cell nuclear transfer (SCNT) is still frustratingly inefficient. Inefficiency manifests itself at different levels, which are currently not well integrated. At the molecular level, it leads to widespread genetic, epigenetic and transcriptional aberrations in cloned embryos. At the organismal level, these genome-wide abnormalities compromise development of cloned foetuses and offspring. Specific molecular defects need to be causally linked to specific cloned phenotypes, in order to design specific treatments to correct them. Cloning efficiency depends on the ability of the nuclear donor cell to be fully reprogrammed into an embryonic state and the ability of the enucleated recipient cell to carry out the reprogramming reactions. It has been postulated that reprogrammability of the somatic donor cell epigenome is influenced by its differentiation status. However, direct comparisons between cells of divergent differentiation status within several somatic lineages have found no conclusive evidence for this. Choosing somatic stem cells as donors has not improved cloning efficiency, indicating that donor cell type may be less critical for cloning success. Different recipient cells, on the other hand, vary in their reprogramming ability. In bovine, using zygotes instead of oocytes has increased cloning success. Other improvements in livestock cloning efficiency include better coordinating donor cell type with cell cycle stage and aggregating cloned embryos. In the future, it will be important to demonstrate if these small increases at every step are cumulative, adding up to an integrated cloning protocol with greatly improved efficiency.

Cloning animals by somatic cell nuclear transfer – biological factors

PubMed Central

Tian, X Cindy; Kubota, Chikara; Enright, Brian; Yang, Xiangzhong

2003-01-01

Cloning by nuclear transfer using mammalian somatic cells has enormous potential application. However, somatic cloning has been inefficient in all species in which live clones have been produced. High abortion and fetal mortality rates are commonly observed. These developmental defects have been attributed to incomplete reprogramming of the somatic nuclei by the cloning process. Various strategies have been used to improve the efficiency of nuclear transfer, however, significant breakthroughs are yet to happen. In this review we will discuss studies conducted, in our laboratories and those of others, to gain a better understanding of nuclear reprogramming. Because cattle are a species widely used for nuclear transfer studies, and more laboratories have succeeded in cloning cattle than any other specie, this review will be focused on somatic cell cloning of cattle. PMID:14614770

Cloning animals by somatic cell nuclear transfer--biological factors.

PubMed

Tian, X Cindy; Kubota, Chikara; Enright, Brian; Yang, Xiangzhong

2003-11-13

Cloning by nuclear transfer using mammalian somatic cells has enormous potential application. However, somatic cloning has been inefficient in all species in which live clones have been produced. High abortion and fetal mortality rates are commonly observed. These developmental defects have been attributed to incomplete reprogramming of the somatic nuclei by the cloning process. Various strategies have been used to improve the efficiency of nuclear transfer, however, significant breakthroughs are yet to happen. In this review we will discuss studies conducted, in our laboratories and those of others, to gain a better understanding of nuclear reprogramming. Because cattle are a species widely used for nuclear transfer studies, and more laboratories have succeeded in cloning cattle than any other species, this review will be focused on somatic cell cloning of cattle.

Somatic Nucleus Reprogramming Is Significantly Improved by m-Carboxycinnamic Acid Bishydroxamide, a Histone Deacetylase Inhibitor*

PubMed Central

Dai, Xiangpeng; Hao, Jie; Hou, Xiao-jun; Hai, Tang; Fan, Yong; Yu, Yang; Jouneau, Alice; Wang, Liu; Zhou, Qi

2010-01-01

Somatic cell nuclear transfer (SCNT) has shown tremendous potential for understanding the mechanisms of reprogramming and creating applications in the realms of agriculture, therapeutics, and regenerative medicine, although the efficiency of reprogramming is still low. Somatic nucleus reprogramming is triggered in the short time after transfer into recipient cytoplasm, and therefore, this period is regarded as a key stage for optimizing SCNT. Here we report that CBHA, a histone deacetylase inhibitor, modifies the acetylation status of somatic nuclei and increases the developmental potential of mouse cloned embryos to reach pre- and post-implantation stages. Furthermore, the cloned embryos treated by CBHA displayed higher efficiency in the derivation of nuclear transfer embryonic stem cell lines by promoting outgrowths. More importantly, CBHA increased blastocyst quality compared with trichostatin A, another prevalent histone deacetylase inhibitor reported previously. Use of CBHA should improve the productivity of SCNT for a variety of research and clinical applications, and comparisons of cells with different levels of pluripotency and treated with CBHA versus trichostatin A will facilitate studies of the mechanisms of reprogramming. PMID:20566633

Efficient modification of the myostatin gene in porcine somatic cells and generation of knockout piglets.

PubMed

Rao, Shengbin; Fujimura, Tatsuya; Matsunari, Hitomi; Sakuma, Tetsushi; Nakano, Kazuaki; Watanabe, Masahito; Asano, Yoshinori; Kitagawa, Eri; Yamamoto, Takashi; Nagashima, Hiroshi

2016-01-01

Myostatin (MSTN) is a negative regulator of myogenesis, and disruption of its function causes increased muscle mass in various species. Here, we report the generation of MSTN-knockout (KO) pigs using genome editing technology combined with somatic-cell nuclear transfer (SCNT). Transcription activator-like effector nuclease (TALEN) with non-repeat-variable di-residue variations, called Platinum TALEN, was highly efficient in modifying genes in porcine somatic cells, which were then used for SCNT to create MSTN KO piglets. These piglets exhibited a double-muscled phenotype, possessing a higher body weight and longissimus muscle mass measuring 170% that of wild-type piglets, with double the number of muscle fibers. These results demonstrate that loss of MSTN increases muscle mass in pigs, which may help increase pork production for consumption in the future. © 2015 Wiley Periodicals, Inc.

Impeding Xist expression from the active X chromosome improves mouse somatic cell nuclear transfer.

PubMed

Inoue, Kimiko; Kohda, Takashi; Sugimoto, Michihiko; Sado, Takashi; Ogonuki, Narumi; Matoba, Shogo; Shiura, Hirosuke; Ikeda, Rieko; Mochida, Keiji; Fujii, Takashi; Sawai, Ken; Otte, Arie P; Tian, X Cindy; Yang, Xiangzhong; Ishino, Fumitoshi; Abe, Kuniya; Ogura, Atsuo

2010-10-22

Cloning mammals by means of somatic cell nuclear transfer (SCNT) is highly inefficient because of erroneous reprogramming of the donor genome. Reprogramming errors appear to arise randomly, but the nature of nonrandom, SCNT-specific errors remains elusive. We found that Xist, a noncoding RNA that inactivates one of the two X chromosomes in females, was ectopically expressed from the active X (Xa) chromosome in cloned mouse embryos of both sexes. Deletion of Xist on Xa showed normal global gene expression and resulted in about an eight- to ninefold increase in cloning efficiency. We also identified an Xist-independent mechanism that specifically down-regulated a subset of X-linked genes through somatic-type repressive histone blocks. Thus, we have identified nonrandom reprogramming errors in mouse cloning that can be altered to improve the efficiency of SCNT methods.

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

Influences of DMP on the Fertilization Process and Subsequent Embryogenesis of Abalone (Haliotis diversicolor supertexta) by Gametes Exposure

PubMed Central

Cai, Zhong-Hua

2011-01-01

Di-methyl phthalate (DMP), a typical endocrine disrupting chemical (EDC), is ubiquitously distributed in aquatic environments; yet studies regarding its impact on gametes and the resulting effects on embryogenesis in marine gastropods are relatively scarce. In this study, the influences of DMP on the gametes and subsequent developmental process of abalone (Haliotis diversicolor supertexta, a representative marine benthic gastropod) were assessed. Newborn abalone eggs and sperm were exposed separately to different DMP concentrations (1, 10 or 100 ppb) for 60 min. At the end-point of exposure, the DMP-treated eggs and sperm were collected for analysis of their ultra-structures, ATPase activities and total lipid levels, and the fertilized gametes (embryos) were collected to monitor related reproductive parameters (fertilization rate, abnormal development rate and hatching success rate). Treatment with DMP did not significantly alter the structure or total lipid content of eggs at any of the doses tested. Hatching failures and morphological abnormalities were only observed with the highest dose of DMP (100 ppb). However, DMP exposure did suppress sperm ATPase activities and affect the morphological character of their mitochondria. DMP-treated sperm exhibited dose-dependent decreases in fertilization efficiency, morphogenesis and hatchability. Relatively obvious toxicological effects were observed when both sperm and eggs were exposed to DMP. Furthermore, RT-PCR results indicate that treatment of gametes with DMP changed the expression patterns of physiologically-regulated genes (cyp3a, 17β-HSD-11 and 17β-HSD-12) in subsequent embryogenesis. Taken together, this study proofed that pre-fertilization exposure of abalone eggs, sperm or both to DMP adversely affects the fertilization process and subsequent embryogenesis. PMID:22028799

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.

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.

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

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.

Efficient embryogenic suspension culturing and rapid transformation of a range of elite genotypes of sweet potato (Ipomoea batatas [L.] Lam.).

PubMed

Yang, Jun; Bi, Hui-Ping; Fan, Wei-Juan; Zhang, Min; Wang, Hong-Xia; Zhang, Peng

2011-12-01

Efficient Agrobacterium tumefaciens-mediated transformation was developed using embryogenic suspension cell cultures of elite sweet potato (Ipomoea batatas [L.] Lam.) cultivars, including Ayamurasaki, Sushu2, Sushu9, Sushu11, Wanshu1, Xushu18 and Xushu22. Embryogenic suspension cultures were established in LCP medium using embryogenic calli induced from apical or axillary buds on an induction medium containing 2 mg l(-1) 2,4-D. Suspension cultures were co-cultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with the hpt gene as a selectable marker and an intron-interrupted uidA gene as a visible marker. Several key steps of the sweet potato transformation system have been investigated and optimized, including the appropriate antibiotics and their concentrations for suppressing Agrobacterium growth and the optimal doses of hygromycin for transformant selection. A total of 485 putative transgenic plant lines were produced from the transformed calli via somatic embryogenesis and germination to plants under 10 mg l(-1) hygromycin and 200 mg l(-1) cefotaxime. PCR, GUS and Southern blot analyses of the regenerated plants showed that 92.35% of them were transgenic. The number of T-DNA insertions varied from one to three in most transgenic plant lines. Plants showed 100% survival when 308 transgenics were transferred to soil in the greenhouse and then to the field. Most of them were morphologically normal, with the production of storage roots after 3 months of cultivation in the greenhouse or fields. The development of such a robust transformation method suitable to a range of sweet potato genotypes not only provides a routine tool for genetic improvement via transgenesis but also allows us to conduct a functional verification of endogenous genes in sweet potato. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Identifying Candidate Reprogramming Genes in Mouse Induced Pluripotent Stem Cells.

PubMed

Gao, Fang; Li, Jingyu; Zhang, Heng; Yang, Xu; An, Tiezhu

2017-08-01

Factor-based induced reprogramming approaches have tremendous potential for human regenerative medicine, but the efficiencies of these approaches are still low. In this study, we analyzed the global transcriptional profiles of mouse induced pluripotent stem cells (miPSCs) and mouse embryonic stem cells (mESCs) from seven different labs and present here the first successful clustering according to cell type, not by lab of origin. We identified 2131 different expression genes (DEs) as candidate pluripotency-associated genes by comparing mESCs/miPSCs with somatic cells and 720 DEs between miPSCs and mESCs. Interestingly, there was a significant overlap between the two DE sets. Therefore, we defined the overlap DEs as "consensus DEs" including 313 miPSC-specific genes expressed at a higher level in miPSCs versus mESCs and 184 mESC-specific genes in total and reasoned that these may contribute to the differences in pluripotency between mESCs and miPSCs. A classification of "consensus DEs" according to their different expression levels between somatic cells and mESCs/miPSCs shows that 86% of the miPSC-specific genes are more highly expressed in somatic cells, while 73% of mESC-specific genes are highly expressed in mESCs/miPSCs, indicating that the miPSCs have not efficiently silenced the expression pattern of the somatic cells from which they are derived and failed to completely induce the genes with high expression levels in mESCs. We further revealed a strong correlation between oocyte-enriched factors and insufficiently induced mESC-specific genes and identified 11 hub genes via network analysis. In light of these findings, we postulated that these key hub genes might not only drive somatic cell nuclear transfer (SCNT) reprogramming but also augment the efficiency and quality of miPSC reprogramming.

Mammalian nuclear transplantation to Germinal Vesicle stage Xenopus oocytes – A method for quantitative transcriptional reprogramming

PubMed Central

Halley-Stott, R.P.; Pasque, V.; Astrand, C.; Miyamoto, K.; Simeoni, I.; Jullien, J.; Gurdon, J.B.

2010-01-01

Full-grown Xenopus oocytes in first meiotic prophase contain an immensely enlarged nucleus, the Germinal Vesicle (GV), that can be injected with several hundred somatic cell nuclei. When the nuclei of mammalian somatic cells or cultured cell lines are injected into a GV, a wide range of genes that are not transcribed in the donor cells, including pluripotency genes, start to be transcriptionally activated, and synthesize primary transcripts continuously for several days. Because of the large size and abundance of Xenopus laevis oocytes, this experimental system offers an opportunity to understand the mechanisms by which somatic cell nuclei can be reprogrammed to transcribe genes characteristic of oocytes and early embryos. The use of mammalian nuclei ensures that there is no background of endogenous maternal transcripts of the kind that are induced. The induced gene transcription takes place in the absence of cell division or DNA synthesis and does not require protein synthesis. Here we summarize new as well as established results that characterize this experimental system. In particular, we describe optimal conditions for transplanting somatic nuclei to oocytes and for the efficient activation of transcription by transplanted nuclei. We make a quantitative determination of transcript numbers for pluripotency and housekeeping genes, comparing cultured somatic cell nuclei with those of embryonic stem cells. Surprisingly we find that the transcriptional activation of somatic nuclei differs substantially from one donor cell-type to another and in respect of different pluripotency genes. We also determine the efficiency of an injected mRNA translation into protein. PMID:20123126

Bovine somatic cell nuclear transfer.

PubMed

Ross, Pablo J; Cibelli, Jose B

2010-01-01

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

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.

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.

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.

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

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.

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.

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.

Single inverted terminal repeats of the Junonia coenia Densovirus promotes somatic chromosomal integration of vector plasmids in insect cells and supports high efficiency expression

USDA-ARS?s Scientific Manuscript database

Plasmids that contain a disrupted genome of the Junonia coenia densovirus (JcDNV) integrate into the chromosomes of the somatic cells of insects. When subcloned individually, both the P9 inverted terminal repeat (P9-ITR) and the P93-ITR promote the chromosomal integration of vector plasmids in insec...

Mismatch repair genes Mlh1 and Mlh3 modify CAG instability in Huntington's disease mice: genome-wide and candidate approaches.

PubMed

Pinto, Ricardo Mouro; Dragileva, Ella; Kirby, Andrew; Lloret, Alejandro; Lopez, Edith; St Claire, Jason; Panigrahi, Gagan B; Hou, Caixia; Holloway, Kim; Gillis, Tammy; Guide, Jolene R; Cohen, Paula E; Li, Guo-Min; Pearson, Christopher E; Daly, Mark J; Wheeler, Vanessa C

2013-10-01

The Huntington's disease gene (HTT) CAG repeat mutation undergoes somatic expansion that correlates with pathogenesis. Modifiers of somatic expansion may therefore provide routes for therapies targeting the underlying mutation, an approach that is likely applicable to other trinucleotide repeat diseases. Huntington's disease Hdh(Q111) mice exhibit higher levels of somatic HTT CAG expansion on a C57BL/6 genetic background (B6.Hdh(Q111) ) than on a 129 background (129.Hdh(Q111) ). Linkage mapping in (B6x129).Hdh(Q111) F2 intercross animals identified a single quantitative trait locus underlying the strain-specific difference in expansion in the striatum, implicating mismatch repair (MMR) gene Mlh1 as the most likely candidate modifier. Crossing B6.Hdh(Q111) mice onto an Mlh1 null background demonstrated that Mlh1 is essential for somatic CAG expansions and that it is an enhancer of nuclear huntingtin accumulation in striatal neurons. Hdh(Q111) somatic expansion was also abolished in mice deficient in the Mlh3 gene, implicating MutLγ (MLH1-MLH3) complex as a key driver of somatic expansion. Strikingly, Mlh1 and Mlh3 genes encoding MMR effector proteins were as critical to somatic expansion as Msh2 and Msh3 genes encoding DNA mismatch recognition complex MutSβ (MSH2-MSH3). The Mlh1 locus is highly polymorphic between B6 and 129 strains. While we were unable to detect any difference in base-base mismatch or short slipped-repeat repair activity between B6 and 129 MLH1 variants, repair efficiency was MLH1 dose-dependent. MLH1 mRNA and protein levels were significantly decreased in 129 mice compared to B6 mice, consistent with a dose-sensitive MLH1-dependent DNA repair mechanism underlying the somatic expansion difference between these strains. Together, these data identify Mlh1 and Mlh3 as novel critical genetic modifiers of HTT CAG instability, point to Mlh1 genetic variation as the likely source of the instability difference in B6 and 129 strains and suggest that MLH1 protein levels play an important role in driving of the efficiency of somatic expansions.

Mismatch Repair Genes Mlh1 and Mlh3 Modify CAG Instability in Huntington's Disease Mice: Genome-Wide and Candidate Approaches

PubMed Central

Pinto, Ricardo Mouro; Dragileva, Ella; Kirby, Andrew; Lloret, Alejandro; Lopez, Edith; St. Claire, Jason; Panigrahi, Gagan B.; Hou, Caixia; Holloway, Kim; Gillis, Tammy; Guide, Jolene R.; Cohen, Paula E.; Li, Guo-Min; Pearson, Christopher E.; Daly, Mark J.; Wheeler, Vanessa C.

2013-01-01

The Huntington's disease gene (HTT) CAG repeat mutation undergoes somatic expansion that correlates with pathogenesis. Modifiers of somatic expansion may therefore provide routes for therapies targeting the underlying mutation, an approach that is likely applicable to other trinucleotide repeat diseases. Huntington's disease HdhQ111 mice exhibit higher levels of somatic HTT CAG expansion on a C57BL/6 genetic background (B6.HdhQ111) than on a 129 background (129.HdhQ111). Linkage mapping in (B6x129).HdhQ111 F2 intercross animals identified a single quantitative trait locus underlying the strain-specific difference in expansion in the striatum, implicating mismatch repair (MMR) gene Mlh1 as the most likely candidate modifier. Crossing B6.HdhQ111 mice onto an Mlh1 null background demonstrated that Mlh1 is essential for somatic CAG expansions and that it is an enhancer of nuclear huntingtin accumulation in striatal neurons. HdhQ111 somatic expansion was also abolished in mice deficient in the Mlh3 gene, implicating MutLγ (MLH1–MLH3) complex as a key driver of somatic expansion. Strikingly, Mlh1 and Mlh3 genes encoding MMR effector proteins were as critical to somatic expansion as Msh2 and Msh3 genes encoding DNA mismatch recognition complex MutSβ (MSH2–MSH3). The Mlh1 locus is highly polymorphic between B6 and 129 strains. While we were unable to detect any difference in base-base mismatch or short slipped-repeat repair activity between B6 and 129 MLH1 variants, repair efficiency was MLH1 dose-dependent. MLH1 mRNA and protein levels were significantly decreased in 129 mice compared to B6 mice, consistent with a dose-sensitive MLH1-dependent DNA repair mechanism underlying the somatic expansion difference between these strains. Together, these data identify Mlh1 and Mlh3 as novel critical genetic modifiers of HTT CAG instability, point to Mlh1 genetic variation as the likely source of the instability difference in B6 and 129 strains and suggest that MLH1 protein levels play an important role in driving of the efficiency of somatic expansions. PMID:24204323

Overexpression of Tet3 in donor cells enhances goat somatic cell nuclear transfer efficiency.

PubMed

Han, Chengquan; Deng, Ruizhi; Mao, Tingchao; Luo, Yan; Wei, Biao; Meng, Peng; Zhao, Lu; Zhang, Qing; Quan, Fusheng; Liu, Jun; Zhang, Yong

2018-05-23

Ten-eleven translocation 3 (TET3) mediates active DNA demethylation of paternal genomes during mouse embryonic development. However, the mechanism of DNA demethylation in goat embryos remains unknown. In addition, aberrant DNA methylation reprogramming prevalently occurs in embryos cloned by somatic cell nuclear transfer (SCNT). In this study, we reported that TET3 is a key factor in DNA demethylation in goat pre-implantation embryos. Knockdown of Tet3 hindered DNA demethylation at the two- to four-cell stage in goat embryos and decreased Nanog expression in blastocysts. Overexpression of Tet3 in somatic cells can initiate DNA demethylation, reduce 5-methylcytosine level, increase 5-hydroxymethylcytosine level and promote the expression of key pluripotency genes. After SCNT, overexpression of Tet3 in donor cells corrected abnormal DNA hypermethylation of cloned embryos and significantly enhanced in vitro and in vivo developmental rate (P < 0.05). We conclude that overexpression of Tet3 in donor cells significantly improves goat SCNT efficiency. © 2018 Federation of European Biochemical Societies.

Role of biotechnological interventions in the improvement of castor (Ricinus communis L.) and Jatropha curcas L.

PubMed

Sujatha, M; Reddy, T P; Mahasi, M J

2008-01-01

Castor and Jatropha belong to the Euphorbiaceae family. This review highlights the role of biotechnological tools in the genetic improvement of castor and jatropha. Castor is monotypic and breeding programmes have mostly relied on the variability available in the primary gene pool. The major constraints limiting profitable cultivation are: vulnerability to insect pests and diseases, and the press cake is toxic which restrict its use as cattle feed. Conventional breeding techniques have limited scope in improvement of resistance to biotic stresses and in quality improvement owing to low genetic variability for these traits. Genetic diversity was assessed using protein based markers while use of molecular markers is at infancy. In vitro studies in castor have been successful in shoot proliferation from meristematic explants, but not callus-mediated regeneration. Genetic transformation experiments have been initiated for development of insect resistant and ricin-free transgenics with very low transformation frequency. In tropical and subtropical countries jatropha is viewed as a potential biofuel crop. The limitations in available germplasm include; lack of knowledge of the genetic base, poor yields, low genetic diversity and vulnerability to a wide array of insects and diseases. Great scope exists for genetic improvement through conventional methods, induced mutations, interspecific hybridization and genetic transformation. Reliable and highly efficient tissue culture protocols for direct and callus-mediated shoot regeneration and somatic embryogenesis are established for jatropha which indicates potential for widening the genetic base through biotechnological tools. Assessment of genetic diversity using molecular markers disclosed low interaccessional variability in local Jatropha curcas germplasm. The current status and future prospects of in vitro regeneration, genetic transformation and the role of molecular tools in the genetic enhancement of the two-oilseed crops are discussed.

GhL1L1 affects cell fate specification by regulating GhPIN1-mediated auxin distribution.

PubMed

Xu, Jiao; Yang, Xiyan; Li, Baoqi; Chen, Lin; Min, Ling; Zhang, Xianlong

2018-05-13

Auxin is as an efficient initiator and regulator of cell fate during somatic embryogenesis (SE), but the molecular mechanisms and regulating networks of this process are not well understood. In this report, we analysed SE process induced by Leafy cotyledon1-like 1 (GhL1L1), a NF-YB subfamily gene specifically expressed in embryonic tissues in cotton. We also identified the target gene of GhL1L1, and its role in auxin distribution and cell fate specification during embryonic development was analysed. Overexpression of GhL1L1 accelerated embryonic cell formation, associated with an increased concentration of IAA in embryogenic calluses (ECs) and in the shoot apical meristem (SAM), corresponding to altered expression of the auxin transport gene GhPIN1. By contrast, GhL1L1-deficient explants showed retarded embryonic cell formation, and the concentration of IAA was decreased in GhL1L1-deficient ECs. Disruption of auxin distribution accelerated the specification of embryonic cell fate together with regulation of GhPIN1. Furthermore, we showed that PHOSPHATASE 2AA2 (GhPP2AA2) was activated by GhL1L1 through targeting the G-box of its promoter, hence regulating the activity of GhPIN1 protein. Our results indicate that GhL1L1 functions as a key regulator in auxin distribution to regulate cell fate specification in cotton and contribute to the understanding of the complex process of SE in plant species. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Mutalisk: a web-based somatic MUTation AnaLyIS toolKit for genomic, transcriptional and epigenomic signatures.

PubMed

Lee, Jongkeun; Lee, Andy Jinseok; Lee, June-Koo; Park, Jongkeun; Kwon, Youngoh; Park, Seongyeol; Chun, Hyonho; Ju, Young Seok; Hong, Dongwan

2018-05-22

Somatic genome mutations occur due to combinations of various intrinsic/extrinsic mutational processes and DNA repair mechanisms. Different molecular processes frequently generate different signatures of somatic mutations in their own favored contexts. As a result, the regional somatic mutation rate is dependent on the local DNA sequence, the DNA replication/RNA transcription dynamics and epigenomic chromatin organization landscape in the genome. Here, we propose an online computational framework, termed Mutalisk, which correlates somatic mutations with various genomic, transcriptional and epigenomic features in order to understand mutational processes that contribute to the generation of the mutations. This user-friendly tool explores the presence of localized hypermutations (kataegis), dissects the spectrum of mutations into the maximum likelihood combination of known mutational signatures and associates the mutation density with numerous regulatory elements in the genome. As a result, global patterns of somatic mutations in any query sample can be efficiently screened, thus enabling a deeper understanding of various mutagenic factors. This tool will facilitate more effective downstream analyses of cancer genome sequences to elucidate the diversity of mutational processes underlying the development and clonal evolution of cancer cells. Mutalisk is freely available at http://mutalisk.org.

Factors affecting the electrofusion of mouse and ferret oocytes with ferret somatic cells.

PubMed

Li, Ziyi; Sun, Xingshen; Chen, Juan; Leno, Gregory H; Engelhardt, John F

2005-09-01

The domestic ferret, Mustela putorius furos, holds great promise as a genetic model for human lung disease, provided that key technologies for somatic cell nuclear transfer (SCNT) are developed. In this report, we extend our understanding of SCNT in this species by defining conditions for efficient cell fusion by electrical pulse. Two experimental systems were employed in this study. First, in vivo-matured mouse oocytes and ferret somatic cells were used to establish general parameters for fusion. One fibroblast, or cumulus cell, was agglutinated to nucleate, zona pellucida-free, mouse oocytes, and subjected to an electrical pulse. Similar electrical pulse conditions were also tested with 1 or 2 somatic cells inserted into the perivitelline space (PVS) of intact mouse oocytes. The fusion rate for a single fibroblast with a zona-free oocyte was 80.2%, significantly higher (P < 0.05) than that observed for 1, or 2, fibroblasts placed in the PVS (52.0% and 63.8%, respectively). The fusion rate (44.1%) following insertion of two cumulus cells was significantly higher (P < 0.05) than that following insertion of one cumulus cell (25.1%). Second, in vitro-matured ferret oocytes were enucleated, and one to three fibroblasts or cumulus cells were inserted into the PVS. Zona pellucida-free ferret oocytes were fragile and excluded from the study. The fusion rates with two or three fibroblasts were 71.4% and 76.8%, respectively; significantly higher (P < 0.05) than that for one fibroblast (48.6%). This cell number-dependent difference in fusion efficiency was also observed with cumulus cells. Fusion-derived (ferret-ferret) NT embryos cleaved, formed blastocysts in vitro, and underwent early-stage fetal development following embryo transfer. The rate of development was cell type-independent, in contrast to the cell type-dependent differences observed in fusion efficiency. In conclusion, fibroblasts fused more efficiently than cumulus cells and the efficiency of single cell fusions was improved when two or more cells were inserted into the PVS. These studies define conditions for efficient cell fusion with ferret oocytes and should facilitate SCNT and the development of genetically defined animal models in this species.

Trauma-informed schools: Child disaster exposure, community violence and somatic symptoms.

PubMed

Lai, Betty S; Osborne, Melissa C; Lee, NaeHyung; Self-Brown, Shannon; Esnard, Ann-Margaret; Kelley, Mary Lou

2018-06-15

Given the increasing prevalence of natural disasters, trauma-informed school settings should include efficient methods for assessing child health and mental health in post-disaster environments. To develop such methods, factors that contribute to children's vulnerability and key signs of distress reactions after disasters need to be understood. To address these issues, we evaluated pre-disaster community violence exposure as a vulnerability factor for children's post-disaster reactions and somatic symptoms as a key post-disaster outcome. We evaluated 426 children exposed to Hurricane Katrina at two timepoints (3-7 months and 13-17 months post-disaster). Structural equation models evaluated community violence exposure, hurricane exposure, and posttraumatic stress and somatic symptoms. Community violence exposure was associated with increased levels of posttraumatic stress symptoms among disaster-impacted youth, and did not moderate the relationship between disaster exposure and posttraumatic stress symptoms. Posttraumatic stress symptoms were associated with somatic symptoms in the short-term recovery period (3-7 months), but not associated with somatic symptoms during the longer-term recovery period (13-17 months). This study did not include school-level factors, and somatic symptoms were based on parent reports. The study did not include parent functioning information or distinguish between whether somatic symptoms were medical or functional in nature. Post-disaster school-based screeners may need to incorporate questions related to children's past exposure to community violence and their somatic symptoms to provide trauma-informed care for children. Copyright © 2018. Published by Elsevier B.V.

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.

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

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

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. 

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.

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

[Product safety analysis of somatic cell cloned bovine].

PubMed

Hua, Song; Lan, Jie; Song, Yongli; Lu, Chenglong; Zhang, Yong

2010-05-01

Somatic cell cloning (nuclear transfer) is a technique through which the nucleus (DNA) of a somatic cell is transferred into an enucleated oocyte for the generation of a new individual, genetically identical to the somatic cell donor. It could be applied for the enhancement of reproduction rate and the improvement of food products involving quality, yield and nutrition. In recent years, the United States, Japan and Europe as well as other countries announced that meat and milk products made from cloned cattle are safe for human consumption. Yet, cloned animals are faced with a wide range of health problems, with a high death rate and a high incidence of disease. The precise causal mechanisms for the low efficiency of cloning remain unclear. Is it safe that any products from cloned animals were allowed into the food supply? This review focuses on the security of meat, milk and products from cloned cattle based on the available data.

RNA splicing and its connection with other regulatory layers in somatic cell reprogramming.

PubMed

Zavolan, Mihaela; Kanitz, Alexander

2018-06-01

Understanding how cell identity is established and maintained is one of the most exciting challenges of molecular biology today. Recent work has added a conserved layer of RNA splicing and other post-transcriptional regulatory processes to the transcriptional and epigenetic networks already known to cooperate in the establishment and maintenance of cell identity. Here we summarize these findings, highlighting specifically the multitude of splicing factors that can modulate the efficiency of somatic cell reprogramming. Distinct patterns of gene expression dynamics of these factors during reprogramming suggest that further improvements in efficiency could be obtained through optimal timing of overexpression or knockdown of individual regulators. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

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.

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.

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.

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.

Transcript levels of several epigenome regulatory genes in bovine somatic donor cells are not correlated with their cloning efficiency.

PubMed

Zhou, Wenli; Sadeghieh, Sanaz; Abruzzese, Ronald; Uppada, Subhadra; Meredith, Justin; Ohlrichs, Charletta; Broek, Diane; Polejaeva, Irina

2009-09-01

Among many factors that potentially affect somatic cell nuclear transfer (SCNT) embryo development is the donor cell itself. Cloning potentials of somatic donor cells vary greatly, possibly because the cells have different capacities to be reprogrammed by ooplasma. It is therefore intriguing to identify factors that regulate the reprogrammability of somatic donor cells. Gene expression analysis is a widely used tool to investigate underlying mechanisms of various phenotypes. In this study, we conducted a retrospective analysis investigating whether donor cell lines with distinct cloning efficiencies express different levels of genes involved in epigenetic reprogramming including histone deacetylase-1 (HDAC1), -2 (HDAC2); DNA methyltransferase-1 (DNMT1), -3a (DNMT3a),-3b (DNMT3b), and the bovine homolog of yeast sucrose nonfermenting-2 (SNF2L), a SWI/SNF family of ATPases. Cell samples from 12 bovine donor cell lines were collected at the time of nuclear transfer experiments and expression levels of the genes were measured using quantitative polymerase chain reaction (PCR). Our results show that there are no significant differences in expression levels of these genes between donor cell lines of high and low cloning efficiency defined as live calving rates, although inverse correlations are observed between in vitro embryo developmental rates and expression levels of HDAC2 and SNF2L. We also show that selection of stable reference genes is important for relative quantification, and different batches of cells can have different gene expression patterns. In summary, we demonstrate that expression levels of these epigenome regulatory genes in bovine donor cells are not correlated with cloning potential. The experimental design and data analysis method reported here can be applied to study any genes expressed in donor cells.

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.

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.

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.

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.

Current reprogramming systems in regenerative medicine: from somatic cells to induced pluripotent stem cells.

PubMed

Hu, Chenxia; Li, Lanjuan

2016-01-01

Induced pluripotent stem cells (iPSCs) paved the way for research fields including cell therapy, drug screening, disease modeling and the mechanism of embryonic development. Although iPSC technology has been improved by various delivery systems, direct transduction and small molecule regulation, low reprogramming efficiency and genomic modification steps still inhibit its clinical use. Improvements in current vectors and the exploration of novel vectors are required to balance efficiency and genomic modification for reprogramming. Herein, we set out a comprehensive analysis of current reprogramming systems for the generation of iPSCs from somatic cells. By clarifying advantages and disadvantages of the current reprogramming systems, we are striding toward an effective route to generate clinical grade iPSCs.

Allotetraploid hybrids between citrus and seven related genera produced by somatic hybridization.

PubMed

Grosser, J W; Mourao-Fo, F A; Gmitter, F G; Louzada, E S; Jiang, J; Baergen, K; Quiros, A; Cabasson, C; Schell, J L; Chandler, J L

1996-04-01

We have developed an efficient protoplast-fusion method to produce somatic hybrid allopolyploid plants that combine Citrus with seven related genera, including four that are sexually incompatible. In this paper we report the creation of 18 new allotetraploid hybrids of Citrus, including ten among sexually incompatible related genera, that may have direct cultivar potential as improved citrus rootstocks. All hybrids were confirmed by cytological and RAPD analyses. If fertile, the attributes of these hybrids may be amenable to further genetic manipulation by breeding at the tetraploid level. Wide somatic hybridization of Citrus via protoplast fusion bypasses biological barriers to the natural allopolyploidization of Citrus, and creates new evolutionary opportunities that would be difficult or impossible to achieve by natural or conventional hybridization.

Coordinated metabolic transitions during Drosophila embryogenesis and the onset of aerobic glycolysis.

PubMed

Tennessen, Jason M; Bertagnolli, Nicolas M; Evans, Janelle; Sieber, Matt H; Cox, James; Thummel, Carl S

2014-03-12

Rapidly proliferating cells such as cancer cells and embryonic stem cells rely on a specialized metabolic program known as aerobic glycolysis, which supports biomass production from carbohydrates. The fruit fly Drosophila melanogaster also utilizes aerobic glycolysis to support the rapid growth that occurs during larval development. Here we use singular value decomposition analysis of modENCODE RNA-seq data combined with GC-MS-based metabolomic analysis to analyze the changes in gene expression and metabolism that occur during Drosophila embryogenesis, spanning the onset of aerobic glycolysis. Unexpectedly, we find that the most common pattern of co-expressed genes in embryos includes the global switch to glycolytic gene expression that occurs midway through embryogenesis. In contrast to the canonical aerobic glycolytic pathway, however, which is accompanied by reduced mitochondrial oxidative metabolism, the expression of genes involved in the tricarboxylic cycle (TCA cycle) and the electron transport chain are also upregulated at this time. Mitochondrial activity, however, appears to be attenuated, as embryos exhibit a block in the TCA cycle that results in elevated levels of citrate, isocitrate, and α-ketoglutarate. We also find that genes involved in lipid breakdown and β-oxidation are upregulated prior to the transcriptional initiation of glycolysis, but are downregulated before the onset of larval development, revealing coordinated use of lipids and carbohydrates during development. These observations demonstrate the efficient use of nutrient stores to support embryonic development, define sequential metabolic transitions during this stage, and demonstrate striking similarities between the metabolic state of late-stage fly embryos and tumor cells. Copyright © 2014 Tennessen et al.

Spatiotemporal coupling and decoupling of gene transcription with DNA replication origins during embryogenesis in C. elegans

PubMed Central

Pourkarimi, Ehsan; Bellush, James M; Whitehouse, Iestyn

2016-01-01

The primary task of developing embryos is genome replication, yet how DNA replication is integrated with the profound cellular changes that occur through development is largely unknown. Using an approach to map DNA replication at high resolution in C. elegans, we show that replication origins are marked with specific histone modifications that define gene enhancers. We demonstrate that the level of enhancer associated modifications scale with the efficiency at which the origin is utilized. By mapping replication origins at different developmental stages, we show that the positions and activity of origins is largely invariant through embryogenesis. Contrary to expectation, we find that replication origins are specified prior to the broad onset of zygotic transcription, yet when transcription initiates it does so in close proximity to the pre-defined replication origins. Transcription and DNA replication origins are correlated, but the association breaks down when embryonic cell division ceases. Collectively, our data indicate that replication origins are fundamental organizers and regulators of gene activity through embryonic development. DOI: http://dx.doi.org/10.7554/eLife.21728.001 PMID:28009254

NetNorM: Capturing cancer-relevant information in somatic exome mutation data with gene networks for cancer stratification and prognosis.

PubMed

Le Morvan, Marine; Zinovyev, Andrei; Vert, Jean-Philippe

2017-06-01

Genome-wide somatic mutation profiles of tumours can now be assessed efficiently and promise to move precision medicine forward. Statistical analysis of mutation profiles is however challenging due to the low frequency of most mutations, the varying mutation rates across tumours, and the presence of a majority of passenger events that hide the contribution of driver events. Here we propose a method, NetNorM, to represent whole-exome somatic mutation data in a form that enhances cancer-relevant information using a gene network as background knowledge. We evaluate its relevance for two tasks: survival prediction and unsupervised patient stratification. Using data from 8 cancer types from The Cancer Genome Atlas (TCGA), we show that it improves over the raw binary mutation data and network diffusion for these two tasks. In doing so, we also provide a thorough assessment of somatic mutations prognostic power which has been overlooked by previous studies because of the sparse and binary nature of mutations.

NetNorM: Capturing cancer-relevant information in somatic exome mutation data with gene networks for cancer stratification and prognosis

PubMed Central

2017-01-01

Genome-wide somatic mutation profiles of tumours can now be assessed efficiently and promise to move precision medicine forward. Statistical analysis of mutation profiles is however challenging due to the low frequency of most mutations, the varying mutation rates across tumours, and the presence of a majority of passenger events that hide the contribution of driver events. Here we propose a method, NetNorM, to represent whole-exome somatic mutation data in a form that enhances cancer-relevant information using a gene network as background knowledge. We evaluate its relevance for two tasks: survival prediction and unsupervised patient stratification. Using data from 8 cancer types from The Cancer Genome Atlas (TCGA), we show that it improves over the raw binary mutation data and network diffusion for these two tasks. In doing so, we also provide a thorough assessment of somatic mutations prognostic power which has been overlooked by previous studies because of the sparse and binary nature of mutations. PMID:28650955

Genetic improvement of mastitis resistance: validation of somatic cell score and clinical mastitis as selection criteria.

PubMed

Odegård, J; Klemetsdal, G; Heringstad, B

2003-12-01

Mean daughter deviations for clinical mastitis among second-crop daughters were regressed on predicted transmitting abilities for clinical mastitis and lactation mean somatic cell score in first-crop daughters to validate the predictive ability of these traits as selection criteria for reduced incidence of clinical mastitis. A total of 321 sires had 684,897 second-crop daughters, while predicted transmitting abilities were calculated for 2159 sires, based on 495,681 records of first-crop daughters. Predictive ability, as a measure of efficiency of selection, was 23 to 43% higher for clinical mastitis than for lactation mean somatic cell score. Compared to single-trait selection, predictive ability improved 8 to 13% from utilizing information on both traits. The relative weight that should be assigned to standardized predicted transmitting abilities from univariate genetic analyses were 60 to 67% for clinical mastitis and 33 to 40% for lactation mean somatic cell score. No significant nonlinear genetic relationship between the two traits was found.

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.

[Nuclear transfer of goat somatic cells transgenic for human lactoferrin].

PubMed

Li, Lan; Shen, Wei; Pan, Qing-Yu; Min, Ling-Jiang; Sun, Yu-Jiang; Fang, Yong-Wei; Deng, Ji-Xian; Pan, Qing-Jie

2006-12-01

Transgenic animal mammary gland bioreactors are being used to produce recombinant proteins with appropriate post-translational modifications, and nuclear transfer of transgenic somatic cells is a more powerful method to produce mammary gland bioreactor. Here we describe efficient gene transfer and nuclear transfer in goat somatic cells. Gene targeting vector pGBC2LF was constructed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene, and the endogenous start condon was replaced by that of human LF gene. Goat fetal fibroblasts were transfected with linearized pGBC2LF and 14 cell lines were positive according to PCR and Southern blot. The transgenic cells were used as donor cells of nuclear transfer, and some of reconstructed embryos could develop to blastocyst in vitro.

[Efficiency of autogenous training in medical rehabilitation of patients with irritable colon syndrome with constipation dominance].

PubMed

Pakhomova, I V; Aĭvazian, T A; Zaĭtsev, V P; Gusakova, E V; Molina, L P

2008-01-01

It was established that use of autogenous training makes possible to increase efficiency of the therapy, leading to considerable more evident improvement of somatic and psychotic state, decrease of pain syndrome. Predictors of efficiency of autogenous training were marked out. Indications for use the method in medical rehabilitation of patients with irritable colon syndrome with constipation dominance were elaborated.

Recent progress in bovine somatic cell nuclear transfer.

PubMed

Akagi, Satoshi; Geshi, Masaya; Nagai, Takashi

2013-03-01

Bovine somatic cell nuclear transfer (SCNT) embryos can develop to the blastocyst stage at a rate similar to that of embryos produced by in vitro fertilization. However, the full-term developmental rate of SCNT embryos is very low, owing to the high embryonic and fetal losses after embryo transfer. In addition, increased birth weight and postnatal mortality are observed at high rates in cloned calves. The low efficiency of SCNT is probably attributed to incomplete reprogramming of the donor nucleus and most of the developmental problems of clones are thought to be caused by epigenetic defects. Applications of SCNT will depend on improvement in the efficiency of production of healthy cloned calves. In this review, we discuss problems and recent progress in bovine SCNT. © 2013 Japanese Society of Animal Science.

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

Functional enucleation of porcine oocytes for somatic cell nuclear transfer using femtosecond laser pulses

NASA Astrophysics Data System (ADS)

Kuetemeyer, K.; Lucas-Hahn, A.; Petersen, B.; Hassel, P.; Lemme, E.; Niemann, H.; Heisterkamp, A.

2010-02-01

Cloning of several mammalian species has been achieved by somatic cell nuclear transfer over the last decade. However, this method still results in very low efficiencies originating from biological and technical aspects. The highly-invasive mechanical enucleation belongs to the technical aspects and requires considerable micromanipulation skill. In this paper, we present a novel non-invasive method for combined oocyte imaging and automated functional enucleation using femtosecond (fs) laser pulses. After three-dimensional imaging of Hoechst-labeled porcine oocytes by multiphoton microscopy, our self-developed software automatically determined the metaphase plate position and shape. Subsequent irradiation of this volume with the very same laser at higher pulse energies in the low-density-plasma regime was used for metaphase plate ablation. We show that functional fs laser-based enucleation of porcine oocytes completely inhibited further embryonic development while maintaining intact oocyte morphology. In contrast, non-irradiated oocytes were able to develop to the blastocyst stage without significant differences to control oocytes. Our results indicate that fs laser systems offer great potential for oocyte imaging and enucleation as a fast, easy to use and reliable tool which may improve the efficiency of somatic cell clone production.

Ontogeny of plants under various gravity condition

NASA Astrophysics Data System (ADS)

Laurinavičius, R.; Švegždienṡ, D.; Raklevičienė, D.; Kenstavičienė, P.

2001-01-01

The results of experiments performed under conditions of microgravity (MG) or under its simulation on the horizontal clinostat (HC) with the callus, seedlings of various species and embryogenic structures have revealed a definite role of gravity as an ecological factor in the processes of cytomorphogenesis, growth, and development. The transformation of differentiated somatic cells of arabidopsis seed into undifferentiated callus was not inhibited under MG, though modifications of the whole callus morphology and of mean cell and nucleus size were observed. The morphogenesis of polar structures such as root-hair bearing cells of Lactuca primary root has been shown to be modified in the course of differentiation under mass acceleration diminished below 0.1 g. Seed germination and seedling morphogenesis under MG follow their normal course, but a significant stimulation of shoot growth with no effect on primary root growth has been determined. A successful in vitro regeneration of Nicotiana tabacum plantlets from leaf cells and subsequent formation of shoots and roots on a continuously rotating HC as well as the formation of viable seeds during seed-to-seed growth of Arabidopsis plants under MG have indicated that gravity plays but a limited role in the processes of embryogenesis and organogenesis.

Plant cell surface receptor-mediated signaling - a common theme amid diversity.

PubMed

He, Yunxia; Zhou, Jinggeng; Shan, Libo; Meng, Xiangzong

2018-01-29

Sessile plants employ a diverse array of plasma membrane-bound receptors to perceive endogenous and exogenous signals for regulation of plant growth, development and immunity. These cell surface receptors include receptor-like kinases (RLKs) and receptor-like proteins (RLPs) that harbor different extracellular domains for perception of distinct ligands. Several RLK and RLP signaling pathways converge at the somatic embryogenesis receptor kinases (SERKs), which function as shared co-receptors. A repertoire of receptor-like cytoplasmic kinases (RLCKs) associate with the receptor complexes to relay intracellular signaling. Downstream of the receptor complexes, mitogen-activated protein kinase (MAPK) cascades are among the key signaling modules at which the signals converge, and these cascades regulate diverse cellular and physiological responses through phosphorylation of different downstream substrates. In this Review, we summarize the emerging common theme that underlies cell surface receptor-mediated signaling pathways in Arabidopsis thaliana : the dynamic association of RLKs and RLPs with specific co-receptors and RLCKs for signal transduction. We further discuss how signaling specificities are maintained through modules at which signals converge, with a focus on SERK-mediated receptor signaling. © 2018. Published by The Company of Biologists Ltd.

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.

Carboxy-terminal truncation activates glp-1 protein to specify vulval fates in Caenorhabditis elegans.

PubMed

Mango, S E; Maine, E M; Kimble, J

1991-08-29

The glp-1 and lin-12 genes encode homologous transmembrane proteins that may act as receptors for cell interactions during development. The glp-1 product is required for induction of germ-line proliferation and for embryogenesis. By contrast, lin-12 mediates somatic cell interactions, including those between the precursor cells that form the vulval hypodermis (VPCs). Here we analyse an unusual allele of glp-1, glp-1(q35), which displays a semidominant multivulva phenotype (Muv), as well as the typical recessive, loss-of-function Glp phenotypes (sterility and embryonic lethality). We find that the effects of glp-1(q35) on VPC development mimic those of dominant lin-12 mutations, even in the absence of lin-12 activity. The glp-1(q35) gene bears a nonsense mutation predicted to eliminate the 122 C-terminal amino acids, including a ProGluSerThr (PEST) sequence thought to destabilize proteins. We suggest that the carboxy terminus bears a negative regulatory domain which normally inactivates glp-1 in the VPCs. We propose that inappropriate glp-1(q35) activity can substitute for lin-12 to determine vulval fate, perhaps by driving the VPCs to proliferate.

The role of activated charcoal in plant tissue culture.

PubMed

Thomas, T Dennis

2008-01-01

Activated charcoal has a very fine network of pores with large inner surface area on which many substances can be adsorbed. Activated charcoal is often used in tissue culture to improve cell growth and development. It plays a critical role in micropropagation, orchid seed germination, somatic embryogenesis, anther culture, synthetic seed production, protoplast culture, rooting, stem elongation, bulb formation etc. The promotary effects of AC on morphogenesis may be mainly due to its irreversible adsorption of inhibitory compounds in the culture medium and substancially decreasing the toxic metabolites, phenolic exudation and brown exudate accumulation. In addition to this activated charcoal is involved in a number of stimulatory and inhibitory activities including the release of substances naturally present in AC which promote growth, alteration and darkening of culture media, and adsorption of vitamins, metal ions and plant growth regulators, including abscisic acid and gaseous ethylene. The effect of AC on growth regulator uptake is still unclear but some workers believe that AC may gradually release certain adsorbed products, such as nutrients and growth regulators which become available to plants. This review focuses on the various roles of activated charcoal in plant tissue culture and the recent developments in this area.

Hydra, a fruitful model system for 270 years.

PubMed

Galliot, Brigitte

2012-01-01

The discovery of Hydra regeneration by Abraham Trembley in 1744 promoted much scientific curiosity thanks to his clever design of experimental strategies away from the natural environment. Since then, this little freshwater cnidarian polyp flourished as a potent and fruitful model system. Here, we review some general biological questions that benefitted from Hydra research, such as the nature of embryogenesis, neurogenesis, induction by organizers, sex reversal, symbiosis, aging, feeding behavior, light regulation, multipotency of somatic stem cells, temperature-induced cell death, neuronal transdifferentiation, to cite only a few. To understand how phenotypes arise, theoricists also chose Hydra to model patterning and morphogenetic events, providing helpful concepts such as reaction-diffusion, positional information, and autocatalysis combined with lateral inhibition. Indeed, throughout these past 270 years, scientists used transplantation and grafting experiments, together with tissue, cell and molecular labelings, as well as biochemical procedures, in order to establish the solid foundations of cell and developmental biology. Nowadays, thanks to transgenic, genomic and proteomic tools, Hydra remains a promising model for these fields, but also for addressing novel questions such as evolutionary mechanisms, maintenance of dynamic homeostasis, regulation of stemness, functions of autophagy, cell death, stress response, innate immunity, bioactive compounds in ecosystems, ecotoxicant sensing and science communication.

Nucleosomal chromatin in the mature sperm of Drosophila melanogaster.

PubMed

Elnfati, Abdul Hakim; Iles, David; Miller, David

2016-03-01

During spermiogenesis in mammals and many other vertebrate classes, histone-containing nucleosomes are replaced by protamine toroids, which can repackage chromatin at a 10 to 20-fold higher density than in a typical somatic nucleus. However, recent evidence suggests that sperm of many species, including human and mouse retain a small compartment of nucleosomal chromatin, particularly near genes important for embryogenesis. As in mammals, spermiogenesis in the fruit fly, Drosophila melanogaster has also been shown to undergo a programmed substitution of nucleosomes with protamine-like proteins. Using chromatin immunoprecipitation (ChIP) and whole-genome tiling array hybridization (ChIP-chip), supported by immunocytochemical evidence, we show that in a manner analogous to nucleosomal chromatin retention in mammalian spermatozoa, distinct domains packaged by the canonical histones H2A, H2B, H3 and H4 are present in the fly sperm nucleus. We also find evidence for the retention of nucleosomes with specific histone H3 trimethylation marks characteristic of chromatin repression (H3K9me3, H3K27me3) and active transcription (H3K36me3). Raw and processed data from the experiments are available at GEO, accession GSE52165.

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

Germ-line epigenetic modification of the murine Avy allele by nutritional supplementation

PubMed Central

Cropley, Jennifer E.; Suter, Catherine M.; Beckman, Kenneth B.; Martin, David I. K.

2006-01-01

Environmental effects on phenotype can be mediated by epigenetic modifications. The epigenetic state of the murine Avy allele is highly variable, and determines phenotypic effects that vary in a mosaic spectrum that can be shifted by in utero exposure to methyl donor supplementation. We have asked if methyl donor supplementation affects the germ-line epigenetic state of the Avy allele. We find that the somatic epigenetic state of Avy is affected by in utero methyl donor supplementation only when the allele is paternally contributed. Exposure to methyl donor supplementation during midgestation shifts Avy phenotypes not only in the mice exposed as fetuses, but in their offspring. This finding indicates that methyl donors can change the epigenetic state of the Avy allele in the germ line, and that the altered state is retained through the epigenetic resetting that takes place in gametogenesis and embryogenesis. Thus a mother's diet may have an enduring influence on succeeding generations, independent of later changes in diet. Although other reports have suggested such heritable epigenetic changes, this study demonstrates that a specific mammalian gene can be subjected to germ-line epigenetic change. PMID:17101998

Germ-line epigenetic modification of the murine A vy allele by nutritional supplementation.

PubMed

Cropley, Jennifer E; Suter, Catherine M; Beckman, Kenneth B; Martin, David I K

2006-11-14

Environmental effects on phenotype can be mediated by epigenetic modifications. The epigenetic state of the murine A vy allele is highly variable, and determines phenotypic effects that vary in a mosaic spectrum that can be shifted by in utero exposure to methyl donor supplementation. We have asked if methyl donor supplementation affects the germ-line epigenetic state of the A vy allele. We find that the somatic epigenetic state of A vy is affected by in utero methyl donor supplementation only when the allele is paternally contributed. Exposure to methyl donor supplementation during midgestation shifts A vy phenotypes not only in the mice exposed as fetuses, but in their offspring. This finding indicates that methyl donors can change the epigenetic state of the A vy allele in the germ line, and that the altered state is retained through the epigenetic resetting that takes place in gametogenesis and embryogenesis. Thus a mother's diet may have an enduring influence on succeeding generations, independent of later changes in diet. Although other reports have suggested such heritable epigenetic changes, this study demonstrates that a specific mammalian gene can be subjected to germ-line epigenetic change.

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.

The Wnt/β-catenin signaling pathway tips the balance between apoptosis and reprograming of cell fusion hybrids.

PubMed

Lluis, Frederic; Pedone, Elisa; Pepe, Stefano; Cosma, Maria Pia

2010-11-01

Cell-cell fusion contributes to cell differentiation and developmental processes. We have previously showed that activation of Wnt/β-catenin enhances somatic cell reprograming after polyethylene glycol (PEG)-mediated fusion. Here, we show that neural stem cells and ESCs can fuse spontaneously in cocultures, although with very low efficiency (about 2%), as the hybrids undergo apoptosis. In contrast, when Wnt/β-catenin signaling is activated in ESCs and leads to accumulation of low amounts of β-catenin in the nucleus, activated ESCs can reprogram somatic cells with very high efficiency after spontaneous fusion. Furthermore, we also show that different levels of β-catenin accumulation in the ESC nuclei can modulate cell proliferation, although in our experimental setting, cell proliferation does not modulate the reprograming efficiency per se. Overall, the present study provides evidence that spontaneous fusion occurs, while the survival of the reprogramed clones is strictly dependent on induction of a Wnt-mediated reprograming pathway. Copyright © 2010 AlphaMed Press.

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

PubMed

Brauckmann, Sabine

2011-12-01

The paper presents selected figures of chick embryogenesis as depicted in the classic studies of Caspar Friedrich Wolff (1734-1794), Christian Heinrich Pander (1794-1865) and Karl Ernst von Baer (1792-1786). My main objective here is (1) to demonstrate how the imagery of Wolff, Pander and Baer attempted to project an image of a 3-dimensional rotating body into static figures on paper by means of linear contours, and (2) to ponder on the efficacy and pervasiveness of dots, lines and arrows for depicting embryogenesis. Copyright © 2011 Elsevier Ltd. All rights reserved.

Embryogenesis-promoting factors in rat serum.

PubMed

Katoh, M; Kimura, R; Shoji, R

1998-06-15

Regarding whole rat embryo cultures in vitro, rat serum as a culture medium is known to support the normal growth of rat embryos in the organogenesis phase. The purpose of the present study was to isolate the embryogenesis-promoting factors from rat serum as a first step in the development of a defined serum-free medium for a whole embryo culture system. Pooled rat serum after heat inactivation was fractionated into three major peaks (frA, containing a region of void volume, frB, and frC) by gel filtration. The 9.5-day rat embryos that were cultivated for 48 hr in essential salt medium containing frB (with a molecular size range of 100-500 kDa) revealed normal growth. Three proteins (27 kDa, 76 kDa, and 190 kDa) that had the embryogenesis-promoting effects were isolated from 3-hr delayed centrifuged rat serum by the ion exchange chromatography. The 76-kDa protein was found to be rat transferrin by immunoblotting. The 27-kDa protein was identified as apo-AI (the major apoprotein of high-density lipoprotein) by immunoblotting. High-density lipoprotein obtained from pooled rat serum by a NaBr density gradient ultracentrifugation was found to have a positive effect on embryogenesis. The 10-kDa protein was also identified as alpha 1-inhibitor 3 by immunoblotting. In addition, the embryogenesis-promoting effect of the fraction containing 27-kDa and 190-kDa proteins declined within a short period of storage at -20 degrees C. This decrease was countered by supplementing its fraction (D-2) with albumin isolated from rat serum. These results in the present study suggest that transferrin, high-density lipoprotein, and alpha 1-inhibitor 3 in rat serum may be embryogenesis-promoting factors, and that albumin appeared to play a role in the embryogenesis of rat embryos in whole embryo cultures.

Somatic cell nuclear transfer cloning: practical applications and current legislation.

PubMed

Niemann, H; Lucas-Hahn, A

2012-08-01

Somatic cloning is emerging as a new biotechnology by which the opportunities arising from the advances in molecular genetics and genome analysis can be implemented in animal breeding. Significant improvements have been made in SCNT protocols in the past years which now allow to embarking on practical applications. The main areas of application of SCNT are: Reproductive cloning, therapeutic cloning and basic research. A great application potential of SCNT based cloning is the production of genetically modified (transgenic) animals. Somatic cell nuclear transfer based transgenic animal production has significant advances over the previously employed microinjection of foreign DNA into pronuclei of zygotes. This cell based transgenesis is compatible with gene targeting and allows both, the addition of a specific gene and the deletion of an endogenous gene. Efficient transgenic animal production provides numerous opportunities for agriculture and biomedicine. Regulatory agencies around the world have agreed that food derived from cloned animals and their offspring is safe and there is no scientific basis for questioning this. Commercial application of somatic cloning within the EU is via the Novel Food regulation EC No. 258/97. Somatic cloning raises novel questions regarding the ethical and moral status of animals and their welfare which has prompted a controversial discussion in Europe which has not yet been resolved. © 2012 Blackwell Verlag GmbH.

Effects of p-chlorophenoxyisobutyric acid, arabinogalactan, and activated charcoal on microspore embryogenesis in kale.

PubMed

Niu, R Q; Zhang, Y; Tong, Y; Liu, Z Y; Wang, Y H; Feng, H

2015-04-27

To improve embryogenesis in microspore cultures of kale (Brassica oleracea L. var. acephala DC.), 6-benzylaminopurine (6-BA), naphthaleneacetic acid (NAA), arabinogalactan (AG), p-chlorophenoxyisobutyric acid (PCIB), and activated charcoal (AC) were added to the medium using four varieties of kale. The results showed that the addition of AG (0.1-0.2 g/L), AC (0.1-0.2 g/L) or a combination of 6-BA (0.1-0.2 mg/L) and NAA (0.1-0.2 mg/L) promoted embryo-genesis. Adding 40 μM PCIB or a combination of 40 μM PCIB and 0.2 g/L AC to NLN-13 medium at pH 5.8 effectively enhanced embryogenesis. Treatment with a combination of 40 μM PCIB and 10 mg/L AG gave the highest rate of embryonic induction, especially in genotype "Y007," which showed a twelve-fold increase in yield.

Germ cell regeneration-mediated, enhanced mutagenesis in the ascidian Ciona intestinalis reveals flexible germ cell formation from different somatic cells.

PubMed

Yoshida, Keita; Hozumi, Akiko; Treen, Nicholas; Sakuma, Tetsushi; Yamamoto, Takashi; Shirae-Kurabayashi, Maki; Sasakura, Yasunori

2017-03-15

The ascidian Ciona intestinalis has a high regeneration capacity that enables the regeneration of artificially removed primordial germ cells (PGCs) from somatic cells. We utilized PGC regeneration to establish efficient methods of germ line mutagenesis with transcription activator-like effector nucleases (TALENs). When PGCs were artificially removed from animals in which a TALEN pair was expressed, somatic cells harboring mutations in the target gene were converted into germ cells, this germ cell population exhibited higher mutation rates than animals not subjected to PGC removal. PGC regeneration enables us to use TALEN expression vectors of specific somatic tissues for germ cell mutagenesis. Unexpectedly, cis elements for epidermis, neural tissue and muscle could be used for germ cell mutagenesis, indicating there are multiple sources of regenerated PGCs, suggesting a flexibility of differentiated Ciona somatic cells to regain totipotency. Sperm and eggs of a single hermaphroditic, PGC regenerated animal typically have different mutations, suggesting they arise from different cells. PGCs can be generated from somatic cells even though the maternal PGCs are not removed, suggesting that the PGC regeneration is not solely an artificial event but could have an endogenous function in Ciona. This study provides a technical innovation in the genome-editing methods, including easy establishment of mutant lines. Moreover, this study suggests cellular mechanisms and the potential evolutionary significance of PGC regeneration in Ciona. Copyright © 2017 Elsevier Inc. All rights reserved.

Evaluation of porcine stem cell competence for somatic cell nuclear transfer and production of cloned animals.

PubMed

Secher, Jan O; Liu, Ying; Petkov, Stoyan; Luo, Yonglun; Li, Dong; Hall, Vanessa J; Schmidt, Mette; Callesen, Henrik; Bentzon, Jacob F; Sørensen, Charlotte B; Freude, Kristine K; Hyttel, Poul

2017-03-01

Porcine somatic cell nuclear transfer (SCNT) has been used extensively to create genetically modified pigs, but the efficiency of the methodology is still low. It has been hypothesized that pluripotent or multipotent stem cells might result in increased SCNT efficacy as these cells are closer than somatic cells to the epigenetic state found in the blastomeres and therefore need less reprogramming. Our group has worked with porcine SCNT during the last 20 years and here we describe our experience with SCNT of 3 different stem cell lines. The porcine stem cells used were: Induced pluripotent stem cells (iPSCs) created by lentiviral doxycycline-dependent reprogramming and cultered with a GSK3β- and MEK-inhibitor (2i) and leukemia inhibitor factor (LIF) (2i LIF DOX-iPSCs), iPSCs created by a plasmid-based reprogramming and cultured with 2i and fibroblast growth factor (FGF) (2i FGF Pl-iPSCs) and embryonic germ cells (EGCs), which have earlier been characterized as being multipotent. The SCNT efficiencies of these stem cell lines were compared with that of the two fibroblast cell lines from which the iPSC lines were derived. The blastocyst rates for the 2i LIF DOX-iPSCs were 14.7%, for the 2i FGF Pl-iPSC 10.1%, and for the EGCs 34.5% compared with the fibroblast lines yielding 36.7% and 25.2%. The fibroblast- and EGC-derived embryos were used for embryo transfer and produced live offspring at similar low rates of efficiency (3.2 and 4.0%, respectively) and with several instances of malformations. In conclusion, potentially pluripotent porcine stem cells resulted in lower rates of embryonic development upon SCNT than multipotent stem cells and differentiated somatic cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Somatic Cell Nuclear Transfer in the Mouse

NASA Astrophysics Data System (ADS)

Kishigami, Satoshi; Wakayama, Teruhiko

Somatic cell nuclear transfer (SCNT) has become a unique and powerful tool for epigenetic reprogramming research and gene manipulation in animals since “Dolly,” the first animal cloned from an adult cell was reported in 1997. Although the success rates of somatic cloning have been inefficient and the mechanism of reprogramming is still largely unknown, this technique has been proven to work in more than 10 mammalian species. Among them, the mouse provides the best model for both basic and applied research of somatic cloning because of its abounding genetic resources, rapid sexual maturity and propagation, minimal requirements for housing, etc. This chapter describes a basic protocol for mouse cloning using cumulus cells, the most popular cell type for NT, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. In particular, we focus on a new, more efficient mouse cloning protocol using trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, which increases both in vitro and in vivo developmental rates from twofold to fivefold. This new method including TSA will be helpful to establish mouse cloning in many laboratories.

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 final step of the ethylene biosynthesis pathway in turnip tops (Brassica rapa): molecular characterization of the 1-aminocyclopropane-1-carboxylate oxidase BrACO1 throughout zygotic embryogenesis and germination of heterogeneous seeds.

PubMed

Del Carmen Rodríguez-Gacio, María; Nicolás, Carlos; Matilla, Angel Jesús

2004-05-01

In a previous report from the present authors, it was shown that the 1-aminocyclopropane-1-carboxylate (ACC) oxidation may play a crucial role during zygotic embryogenesis of turnip tops seeds. The present study was performed to elucidate the contribution of the silique-wall and seeds in ethylene production during this developmental process. ACC content in the silique wall is only higher than in seeds during the middle phases of zygotic embryogenesis. The ACC-oxidase (ACO) activity peaks in the silique-wall and seeds during the onset of embryogenesis, declining gradually afterwards, being undetectable during desiccation period. Using reverse transcriptase-polymerase chain reaction, one cDNA clone coding for an ACO and called BrACO1, was isolated. The deduced protein for BrACO1 has a molecular weight of 36.8 kDa and a high homology with other crucifer ACOs. The heterologous expression of this cDNA confirmed that BrACO1 is an ACO. The expression of this gene was high during the first phases of silique-wall development, low during the middle phases and undetectable during desiccation. By contrast, BrACO1 transcript was accumulated only in the earliest phases of seed embryogenesis and may participate in the highest ACO activity and ethylene production by seeds at the beginning of embryogenesis. Finally, in this work a correlation between the heterogeneity of Brassica rapa L. cv. Rapa seeds and the ability to oxidize the ACC to ethylene has been demonstrated.

Relations of competitive state anxiety and efficacy of young volleyball players.

PubMed

Milavić, Boris; Jurko, Damir; Grgantov, Zoran

2013-05-01

With the aim of validating the Competitive State Anxiety Inventory on a population of young Croatian volleyball players, 286 examinees, 106 male and 180 female volleyball players (average age of 16.09+/-1.83), filled out the CSAI-2, constructed by Martens, Vealey, Burton, Bump and Smith (1990)1. Given the fact that all scales of the Competitive State Anxiety Inventory have good homogeneity, reliability and sensitivity, it can be concluded that they represent high-quality measuring instruments for measuring psychological characteristics of young volleyball players. Young male and female volleyball players generally have a moderate level of self-confidence, and their cognitive anxiety is more prominent that somatic anxiety. In order to determine the age and gender differences in somatic and cognitive anxiety and self-confidence, parametric analysis of differences was performed and coefficients of the independent samples t-test were calculated. By analysis of differences according to age, it has been established that female junior players, in relation to female youth players, express a significantly lower level of somatic and cognitive anxiety and a significantly higher level of self-confidence. As opposed to female players, male youth and junior players do not differ in any of the analysed variables. By analysis of differences according to gender, it has been established that male youth players have a significantly higher level of self-confidence in comparison to female youth players. No significant differences were found in the level of competitive anxiety and self-confidence by analysis of variance between different player roles. No significant differences were found by discriminant analysis in somatic and cognitive anxiety, and self-confidence of female volleyball players of different situational efficacy. The group of least efficient male volleyball players is characterized by a very low level of self-confidence, while the most efficient group of volleyball players is characterized by a somewhat lower level of cognitive and somatic anxiety.

Production of cloned mice and ES cells from adult somatic cells by nuclear transfer: how to improve cloning efficiency?

PubMed

Wakayama, Teruhiko

2007-02-01

Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), most cloned embryos usually undergo developmental arrest prior to or soon after implantation, and the success rate for producing live offspring by cloning remains below 5%. The low success rate is believed to be associated with epigenetic errors, including abnormal DNA hypermethylation, but the mechanism of "reprogramming" is unclear. We have been able to develop a stable NT method in the mouse in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Especially in the mouse, only a few laboratories can make clones from adult somatic cells, and cloned mice are never successfully produced from most mouse strains. However, this technique promises to be an important tool for future research in basic biology. For example, NT can be used to generate embryonic stem (NT-ES) cell lines from a patient's own somatic cells. We have shown that NT-ES cells are equivalent to ES cells derived from fertilized embryos and that they can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. In general, NT-ES cell techniques are expected to be applied to regenerative medicine; however, this technique can also be applied to the preservation of genetic resources of mouse strain instead of embryos, oocytes and spermatozoa. This review describes how to improve cloning efficiency and NT-ES cell establishment and further applications.

Unravelling polar lipids dynamics during embryonic development of two sympatric brachyuran crabs (Carcinus maenas and Necora puber) using lipidomics

PubMed Central

Rey, Felisa; Alves, Eliana; Melo, Tânia; Domingues, Pedro; Queiroga, Henrique; Rosa, Rui; Domingues, M. Rosário M.; Calado, Ricardo

2015-01-01

Embryogenesis is an important stage of marine invertebrates with bi-phasic life cycles, as it conditions their larval and adult life. Throughout embryogenesis, phospholipids (PL) play a key role as an energy source, as well as constituents of biological membranes. However, the dynamics of PL during embryogenesis in marine invertebrates is still poorly studied. The present work used a lipidomic approach to determine how polar lipid profiles shift during embryogenesis in two sympatric estuarine crabs, Carcinus maenas and Necora puber. The combination of thin layer chromatography, liquid chromatography – mass spectrometry and gas chromatography – mass spectrometry allowed us to achieve an unprecedented resolution on PL classes and molecular species present on newly extruded embryos (stage 1) and those near hatching (stage 3). Embryogenesis proved to be a dynamic process, with four PL classes being recorded in stage 1 embryos (68 molecular species in total) and seven PL classes at stage 3 embryos (98 molecular species in total). The low interspecific difference recorded in the lipidomic profiles of stage 1 embryos appears to indicate the existence of similar maternal investment. The same pattern was recorded for stage 3 embryos revealing a similar catabolism of embryonic resources during incubation for both crab species. PMID:26419891

NvERTx: a gene expression database to compare embryogenesis and regeneration in the sea anemone Nematostella vectensis.

PubMed

Warner, Jacob F; Guerlais, Vincent; Amiel, Aldine R; Johnston, Hereroa; Nedoncelle, Karine; Röttinger, Eric

2018-05-17

For over a century, researchers have been comparing embryogenesis and regeneration hoping that lessons learned from embryonic development will unlock hidden regenerative potential. This problem has historically been a difficult one to investigate because the best regenerative model systems are poor embryonic models and vice versa. Recently, however, there has been renewed interest in this question, as emerging models have allowed researchers to investigate these processes in the same organism. This interest has been further fueled by the advent of high-throughput transcriptomic analyses that provide virtual mountains of data. Here, we present N ematostella vectensis Embryogenesis and Regeneration Transcriptomics (NvERTx), a platform for comparing gene expression during embryogenesis and regeneration. NvERTx consists of close to 50 transcriptomic data sets spanning embryogenesis and regeneration in Nematostella These data were used to perform a robust de novo transcriptome assembly, with which users can search, conduct BLAST analyses, and plot the expression of multiple genes during these two developmental processes. The site is also home to the results of gene clustering analyses, to further mine the data and identify groups of co-expressed genes. The site can be accessed at http://nvertx.kahikai.org. © 2018. Published by The Company of Biologists Ltd.

Effects of donor cell type and genotype on the efficiency of mouse somatic cell cloning.

PubMed

Inoue, Kimiko; Ogonuki, Narumi; Mochida, Keiji; Yamamoto, Yoshie; Takano, Kaoru; Kohda, Takashi; Ishino, Fumitoshi; Ogura, Atsuo

2003-10-01

Although it is widely assumed that the cell type and genotype of the donor cell affect the efficiency of somatic cell cloning, little systematic analysis has been done to verify this assumption. The present study was undertaken to examine whether donor cell type, donor genotype, or a combination thereof increased the efficiency of mouse cloning. Initially we assessed the developmental ability of embryos that were cloned from cumulus or immature Sertoli cells with six different genotypes (i.e., 2 x 6 factorial). Significantly better cleavage rates were obtained with cumulus cells than with Sertoli cells (P < 0.005, two-way ANOVA), which probably was due to the superior cell-cycle synchrony of cumulus cells at G0/G1. After embryo transfer, there was a significant effect of cell type on the birth rate, with Sertoli cells giving the better result (P < 0.005). Furthermore, there was a significant interaction (P < 0.05) between the cell type and genotype, which indicates that cloning efficiency is determined by a combination of these two factors. The highest mean birth rate (10.8 +/- 2.1%) was obtained with (B6 x 129)F1 Sertoli cells. In the second series of experiments, we examined whether the developmental ability of clones with the wild-type genotype (JF1) was improved when combined with the 129 genotype. Normal pups were cloned from cumulus and immature Sertoli cells of the (129 x JF1)F1 and (JF1 x 129)F1 genotypes, whereas no pups were born from cells with the (B6 x JF1)F1 genotype. The present study clearly demonstrates that the efficiency of somatic cell cloning, and in particular fetal survival after embryo transfer, may be improved significantly by choosing the appropriate combinations of cell type and genotype.

Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair

PubMed Central

He, Xiangjun; Tan, Chunlai; Wang, Feng; Wang, Yaofeng; Zhou, Rui; Cui, Dexuan; You, Wenxing; Zhao, Hui; Ren, Jianwei; Feng, Bo

2016-01-01

CRISPR/Cas9-induced site-specific DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. Extensive efforts have been made to knock-in exogenous DNA to a selected genomic locus in human cells; which, however, has focused on HDR-based strategies and was proven inefficient. Here, we report that NHEJ pathway mediates efficient rejoining of genome and plasmids following CRISPR/Cas9-induced DNA DSBs, and promotes high-efficiency DNA integration in various human cell types. With this homology-independent knock-in strategy, integration of a 4.6 kb promoterless ires-eGFP fragment into the GAPDH locus yielded up to 20% GFP+ cells in somatic LO2 cells, and 1.70% GFP+ cells in human embryonic stem cells (ESCs). Quantitative comparison further demonstrated that the NHEJ-based knock-in is more efficient than HDR-mediated gene targeting in all human cell types examined. These data support that CRISPR/Cas9-induced NHEJ provides a valuable new path for efficient genome editing in human ESCs and somatic cells. PMID:26850641

Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair.

PubMed

He, Xiangjun; Tan, Chunlai; Wang, Feng; Wang, Yaofeng; Zhou, Rui; Cui, Dexuan; You, Wenxing; Zhao, Hui; Ren, Jianwei; Feng, Bo

2016-05-19

CRISPR/Cas9-induced site-specific DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. Extensive efforts have been made to knock-in exogenous DNA to a selected genomic locus in human cells; which, however, has focused on HDR-based strategies and was proven inefficient. Here, we report that NHEJ pathway mediates efficient rejoining of genome and plasmids following CRISPR/Cas9-induced DNA DSBs, and promotes high-efficiency DNA integration in various human cell types. With this homology-independent knock-in strategy, integration of a 4.6 kb promoterless ires-eGFP fragment into the GAPDH locus yielded up to 20% GFP+ cells in somatic LO2 cells, and 1.70% GFP+ cells in human embryonic stem cells (ESCs). Quantitative comparison further demonstrated that the NHEJ-based knock-in is more efficient than HDR-mediated gene targeting in all human cell types examined. These data support that CRISPR/Cas9-induced NHEJ provides a valuable new path for efficient genome editing in human ESCs and somatic cells. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Efficient Generation of iPS Cells from Skeletal Muscle Stem Cells

PubMed Central

Tan, Kah Yong; Eminli, Sarah; Hettmer, Simone; Hochedlinger, Konrad; Wagers, Amy J.

2011-01-01

Reprogramming of somatic cells into inducible pluripotent stem cells generally occurs at low efficiency, although what limits reprogramming of particular cell types is poorly understood. Recent data suggest that the differentiation status of the cell targeted for reprogramming may influence its susceptibility to reprogramming as well as the differentiation potential of the induced pluripotent stem (iPS) cells that are derived from it. To assess directly the influence of lineage commitment on iPS cell derivation and differentiation, we evaluated reprogramming in adult stem cell and mature cell populations residing in skeletal muscle. Our data using clonal assays and a second-generation inducible reprogramming system indicate that stem cells found in mouse muscle, including resident satellite cells and mesenchymal progenitors, reprogram with significantly greater efficiency than their more differentiated daughters (myoblasts and fibroblasts). However, in contrast to previous reports, we find no evidence of biased differentiation potential among iPS cells derived from myogenically committed cells. These data support the notion that adult stem cells reprogram more efficiently than terminally differentiated cells, and argue against the suggestion that “epigenetic memory” significantly influences the differentiation potential of iPS cells derived from distinct somatic cell lineages in skeletal muscle. PMID:22028872

Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

PubMed

Burger, Alexa; Lindsay, Helen; Felker, Anastasia; Hess, Christopher; Anders, Carolin; Chiavacci, Elena; Zaugg, Jonas; Weber, Lukas M; Catena, Raul; Jinek, Martin; Robinson, Mark D; Mosimann, Christian

2016-06-01

CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo. © 2016. Published by The Company of Biologists Ltd.

BALSA: integrated secondary analysis for whole-genome and whole-exome sequencing, accelerated by GPU.

PubMed

Luo, Ruibang; Wong, Yiu-Lun; Law, Wai-Chun; Lee, Lap-Kei; Cheung, Jeanno; Liu, Chi-Man; Lam, Tak-Wah

2014-01-01

This paper reports an integrated solution, called BALSA, for the secondary analysis of next generation sequencing data; it exploits the computational power of GPU and an intricate memory management to give a fast and accurate analysis. From raw reads to variants (including SNPs and Indels), BALSA, using just a single computing node with a commodity GPU board, takes 5.5 h to process 50-fold whole genome sequencing (∼750 million 100 bp paired-end reads), or just 25 min for 210-fold whole exome sequencing. BALSA's speed is rooted at its parallel algorithms to effectively exploit a GPU to speed up processes like alignment, realignment and statistical testing. BALSA incorporates a 16-genotype model to support the calling of SNPs and Indels and achieves competitive variant calling accuracy and sensitivity when compared to the ensemble of six popular variant callers. BALSA also supports efficient identification of somatic SNVs and CNVs; experiments showed that BALSA recovers all the previously validated somatic SNVs and CNVs, and it is more sensitive for somatic Indel detection. BALSA outputs variants in VCF format. A pileup-like SNAPSHOT format, while maintaining the same fidelity as BAM in variant calling, enables efficient storage and indexing, and facilitates the App development of downstream analyses. BALSA is available at: http://sourceforge.net/p/balsa.

Cloning cattle: the methods in the madness.

PubMed

Oback, Björn; Wells, David N

2007-01-01

Somatic cell nuclear transfer (SCNT) is much more widely and efficiently practiced in cattle than in any other species, making this arguably the most important mammal cloned to date. While the initial objective behind cattle cloning was commercially driven--in particular to multiply genetically superior animals with desired phenotypic traits and to produce genetically modified animals-researchers have now started to use bovine SCNT as a tool to address diverse questions in developmental and cell biology. In this paper, we review current cattle cloning methodologies and their potential technical or biological pitfalls at any step of the procedure. In doing so, we focus on one methodological parameter, namely donor cell selection. We emphasize the impact of epigenetic and genetic differences between embryonic, germ, and somatic donor cell types on cloning efficiency. Lastly, we discuss adult phenotypes and fitness of cloned cattle and their offspring and illustrate some of the more imminent commercial cattle cloning applications.

[Nuclear transfer and therapeutic cloning].

PubMed

Xu, Xiao-Ming; Lei, An-Min; Hua, Jin-Lian; Dou, Zhong-Ying

2005-03-01

Nuclear transfer and therapeutic cloning have widespread and attractive prospects in animal agriculture and biomedical applications. We reviewed that the quality of oocytes and nuclear reprogramming of somatic donor cells were the main reasons of the common abnormalities in cloned animals and the low efficiency of cloning and showed the problems and outlets in therapeutic cloning, such as some basic problems in nuclear transfer affected clinical applications of therapeutic cloning. Study on isolation and culture of nuclear transfer embryonic stem (ntES) cells and specific differentiation of ntES cells into important functional cells should be emphasized and could enhance the efficiency. Adult stem cells could help to cure some great diseases, but could not replace therapeutic cloning. Ethics also impeded the development of therapeutic cloning. It is necessary to improve many techniques and reinforce the research of some basic theories, then somatic nuclear transfer and therapeutic cloning may apply to agriculture reproduction and benefit to human life better.

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.

A system for the measurement of gene targeting efficiency in human cell lines using an antibiotic resistance-GFP fusion gene.

PubMed

Konishi, Yuko; Karnan, Sivasundaram; Takahashi, Miyuki; Ota, Akinobu; Damdindorj, Lkhagvasuren; Hosokawa, Yoshitaka; Konishi, Hiroyuki

2012-09-01

Gene targeting in a broad range of human somatic cell lines has been hampered by inefficient homologous recombination. To improve this technology and facilitate its widespread application, it is critical to first have a robust and efficient research system for measuring gene targeting efficiency. Here, using a fusion gene consisting of hygromycin B phosphotransferase and 3'-truncated enhanced GFP (HygR-5' EGFP) as a reporter gene, we created a molecular system monitoring the ratio of homologous to random integration (H/R ratio) of targeting vectors into the genome. Cell clones transduced with a reporter vector containing HygR-5' EGFP were efficiently established from two human somatic cell lines. Established HygR-5' EGFP reporter clones retained their capacity to monitor gene targeting efficiency for a longer duration than a conventional reporter system using an unfused 5' EGFP gene. With the HygR-5' EGFP reporter system, we reproduced previous findings of gene targeting frequency being up-regulated by the use of an adeno-associated viral (AAV) backbone, a promoter-trap system, or a longer homology arm in a targeting vector, suggesting that this system accurately monitors H/R ratio. Thus, our HygR-5' EGFP reporter system will assist in the development of an efficient AAV-based gene targeting technology.

Histone h1 depletion impairs embryonic stem cell differentiation.

PubMed

Zhang, Yunzhe; Cooke, Marissa; Panjwani, Shiraj; Cao, Kaixiang; Krauth, Beth; Ho, Po-Yi; Medrzycki, Magdalena; Berhe, Dawit T; Pan, Chenyi; McDevitt, Todd C; Fan, Yuhong

2012-01-01

Pluripotent embryonic stem cells (ESCs) are known to possess a relatively open chromatin structure; yet, despite efforts to characterize the chromatin signatures of ESCs, the role of chromatin compaction in stem cell fate and function remains elusive. Linker histone H1 is important for higher-order chromatin folding and is essential for mammalian embryogenesis. To investigate the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, we examine the differentiation of embryonic stem cells that are depleted of multiple H1 subtypes. H1c/H1d/H1e triple null ESCs are more resistant to spontaneous differentiation in adherent monolayer culture upon removal of leukemia inhibitory factor. Similarly, the majority of the triple-H1 null embryoid bodies (EBs) lack morphological structures representing the three germ layers and retain gene expression signatures characteristic of undifferentiated ESCs. Furthermore, upon neural differentiation of EBs, triple-H1 null cell cultures are deficient in neurite outgrowth and lack efficient activation of neural markers. Finally, we discover that triple-H1 null embryos and EBs fail to fully repress the expression of the pluripotency genes in comparison with wild-type controls and that H1 depletion impairs DNA methylation and changes of histone marks at promoter regions necessary for efficiently silencing pluripotency gene Oct4 during stem cell differentiation and embryogenesis. In summary, we demonstrate that H1 plays a critical role in pluripotent stem cell differentiation, and our results suggest that H1 and chromatin compaction may mediate pluripotent stem cell differentiation through epigenetic repression of the pluripotency genes.

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.

Evaluation of Reference Genes for Quantitative Real-Time PCR in Oil Palm Elite Planting Materials Propagated by Tissue Culture

PubMed Central

Chan, Pek-Lan; Rose, Ray J.; Abdul Murad, Abdul Munir; Zainal, Zamri; Leslie Low, Eng-Ti; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

2014-01-01

Background 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. Results 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. Conclusions 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. PMID:24927412

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

Shoot regeneration and embryogenesis in lily shoot tips cryopreserved by droplet vitrification

USDA-ARS?s Scientific Manuscript database

Shoot regeneration and embryogenesis were, for the first time, achieved directly in shoot tips of Lilium Oriental hybrid ‘Siberia’ following cryopreservation by droplet-vitrification. Shoot tips (2 mm in length) including 2-3 leaf primordia were excised from 4-week-old adventitious shoots directly r...

The thiol compounds glutathione and homoglutathione differentially affect cell development in alfalfa (Medicago sativa L.).

PubMed

Pasternak, Taras; Asard, Han; Potters, Geert; Jansen, Marcel A K

2014-01-01

Glutathione (GSH) is an important scavenger of Reactive Oxygen Species (ROS), precursor of metal chelating phytochelatins, xenobiotic defence compound and regulator of cell proliferation. Homoglutathione (hGSH) is a GSH homologue that is present in several taxa in the family of Fabaceae. It is thought that hGSH performs many of the stress-defence roles typically ascribed to GSH, yet little is known about the potential involvement of hGSH in controlling cell proliferation. Here we show that hGSH/GSH ratios vary across organs and cells and that these changes in hGSH/GSH ratio occur during dedifferentiation and/or cell cycle activation events. The use of a GSH/hGSH biosynthesis inhibitor resulted in impaired cytokinesis in isolated protoplasts, showing the critical importance of these thiol-compounds for cell division. However, exposure of isolated protoplasts to exogenous GSH accelerated cytokinesis, while exogenous hGSH was found to inhibit the same process. We conclude that GSH and hGSH have distinct functional roles in cell cycle regulation in Medicago sativa L. GSH is associated with meristemic cells, and promotes cell cycle activation and induction of somatic embryogenesis, while hGSH is associated with differentiated cells and embryo proliferation. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

An XA21-Associated Kinase (OsSERK2) Regulates Immunity Mediated by the XA21 and XA3 Immune Receptors

PubMed Central

Chen, Xuewei; Zuo, Shimin; Schwessinger, Benjamin; Chern, Mawsheng; Canlas, Patrick E.; Ruan, Deling; Zhou, Xiaogang; Wang, Jing; Daudi, Arsalan; Petzold, Christopher J.; Heazlewood, Joshua L.; Ronald, Pamela C.

2014-01-01

The rice XA21 immune receptor kinase and the structurally related XA3 receptor confer immunity to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight. Here we report the isolation of OsSERK2 (rice somatic embryogenesis receptor kinase 2) and demonstrate that OsSERK2 positively regulates immunity mediated by XA21 and XA3 as well as the rice immune receptor FLS2 (OsFLS2). Rice plants silenced for OsSerk2 display altered morphology and reduced sensitivity to the hormone brassinolide. OsSERK2 interacts with the intracellular domains of each immune receptor in the yeast two-hybrid system in a kinase activity-dependent manner. OsSERK2 undergoes bidirectional transphosphorylation with XA21 in vitro and forms a constitutive complex with XA21 in vivo. These results demonstrate an essential role for OsSERK2 in the function of three rice immune receptors and suggest that direct interaction with the rice immune receptors is critical for their function. Taken together, our findings suggest that the mechanism of OsSERK2-meditated regulation of rice XA21, XA3, and FLS2 differs from that of AtSERK3/BAK1-mediated regulation of Arabidopsis FLS2 and EFR. PMID:24482436

OsSERK1 regulates rice development but not immunity to Xanthomonas oryzae pv. oryzae or Magnaporthe oryzae.

PubMed

Zuo, Shimin; Zhou, Xiaogang; Chen, Mawsheng; Zhang, Shilu; Schwessinger, Benjamin; Ruan, Deling; Yuan, Can; Wang, Jing; Chen, Xuewei; Ronald, Pamela C

2014-12-01

Somatic embryogenesis receptor kinase (SERK) proteins play pivotal roles in regulation of plant development and immunity. The rice genome contains two SERK genes, OsSerk1 and OsSerk2. We previously demonstrated that OsSerk2 is required for rice Xa21-mediated resistance to Xanthomonas oryzae pv. oryzae (Xoo) and for normal development. Here we report the molecular characterization of OsSerk1. Overexpression of OsSerk1 results in a semi-dwarf phenotype whereas silencing of OsSerk1 results in a reduced angle of the lamina joint. OsSerk1 is not required for rice resistance to Xoo or Magnaporthe oryzae. Overexpression of OsSerk1 in OsSerk2-silenced lines complements phenotypes associated with brassinosteroid (BR) signaling defects, but not the disease resistance phenotype mediated by Xa21. In yeast, OsSERK1 interacts with itself forming homodimers, and also interacts with the kinase domains of OsSERK2 and BRI1, respectively. OsSERK1 is a functional protein kinase capable of auto-phosphorylation in vitro. We conclude that, whereas OsSERK2 regulates both rice development and immunity, OsSERK1 functions in rice development but not immunity to Xoo and M. oryzae. © 2014 Institute of Botany, Chinese Academy of Sciences.

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

A highly efficient method for generation of therapeutic quality human pluripotent stem cells by using naive induced pluripotent stem cells nucleus for nuclear transfer.

PubMed

Sanal, Madhusudana Girija

2014-01-01

Even after several years since the discovery of human embryonic stem cells and induced pluripotent stem cells (iPSC), we are still unable to make any significant therapeutic benefits out of them such as cell therapy or generation of organs for transplantation. Recent success in somatic cell nuclear transfer (SCNT) made it possible to generate diploid embryonic stem cells, which opens up the way to make high-quality pluripotent stem cells. However, the process is highly inefficient and hence expensive compared to the generation of iPSC. Even with the latest SCNT technology, we are not sure whether one can make therapeutic quality pluripotent stem cell from any patient's somatic cells or by using oocytes from any donor. Combining iPSC technology with SCNT, that is, by using the nucleus of the candidate somatic cell which got reprogrammed to pluripotent state instead that of the unmodified nucleus of the candidate somatic cell, would boost the efficiency of the technique, and we would be able to generate therapeutic quality pluripotent stem cells. Induced pluripotent stem cell nuclear transfer (iPSCNT) combines the efficiency of iPSC generation with the speed and natural reprogramming environment of SCNT. The new technique may be called iPSCNT. This technique could prove to have very revolutionary benefits for humankind. This could be useful in generating organs for transplantation for patients and for reproductive cloning, especially for childless men and women who cannot have children by any other techniques. When combined with advanced gene editing techniques (such as CRISPR-Cas system) this technique might also prove useful to those who want to have healthy children but suffer from inherited diseases. The current code of ethics may be against reproductive cloning. However, this will change with time as it happened with most of the revolutionary scientific breakthroughs. After all, it is the right of every human to have healthy offspring and it is the question of reproductive freedom and existence.

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

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

PubMed

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

2016-02-01

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

Rosmarinic acid plays a protective role in the embryogenesis of zebrafish exposed to food colours through its influence on aurora kinase A level.

PubMed

Swarnalatha, Y; Jerrine Joseph, I S; Jayakrishna, Tippabathani

2017-05-01

To evaluate the protective nature of the rosmarinic acid from Sphaeranthus amaranthoides during zebra fish embryogenesis. Rosmarinic acid was isolated from the S. amaranthoides. An accurate, sensitive and simple LC-MS analysis was performed to determine the rosmarinic acid from S. amaranthoides. In the present study, zebrafish embryos were exposed to crimson red and sunset yellow at a concentration of 0.1 and 0.5mg/l and the effect of these food colours on the levels of aurora kinase A was studied individually. Aurora kinase A levels are crucial for embryogenesis in zebrafish which is used as model in this study. The decrease of aurora kinase A levels in food colour treated embryos influences the embryogenesis, resulting in short and bent trunk leading to cell death and growth retardation. Elevated levels of aurora kinase A in rosmarinic acid treated groups can be attributed to the restoration of normal growth in zebra fish embryos with well developed brain and eyes. Further insilico docking studies were carried out and target was identified as rosmarinic acid. From the docking studies the docking poses and binding energy confirms that aurora kinase A is the target for rosmarinic acid. Rosmarinic acid was found to play a protective role in the embryogenesis of zebra fish exposed to food colours (crimson red and sunset yellow) through its influence on aurora kinase A levels. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

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

PubMed

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

2016-10-01

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

Ovum pick up, intracytoplasmic sperm injection and somatic cell nuclear transfer in cattle, buffalo and horses: from the research laboratory to clinical practice.

PubMed

Galli, Cesare; Duchi, Roberto; Colleoni, Silvia; Lagutina, Irina; Lazzari, Giovanna

2014-01-01

Assisted reproductive techniques developed for cattle in the last 25 years, like ovum pick up (OPU), intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transfer, have been transferred and adapted to buffalo and horses. The successful clinical applications of these techniques require both the clinical skills specific to each animal species and an experienced laboratory team to support the in vitro phase of the work. In cattle, OPU can be considered a consolidated technology that is rapidly outpacing conventional superovulation for embryo transfer. In buffalo, OPU represents the only possibility for embryo production to advance the implementation of embryo-based biotechnologies in that industry, although it is still mainly in the developmental phase. In the horse, OPU is now an established procedure for breeding from infertile and sporting mares throughout the year. It requires ICSI that in the horse, contrary to what happens in cattle and buffalo, is very efficient and the only option because conventional IVF does not work. Somatic cell nuclear transfer is destined to fill a very small niche for generating animals of extremely high commercial value. The efficiency is low, but because normal animals can be generated it is likely that advancing our knowledge in that field might improve the technology and reduce its cost. Copyright © 2014 Elsevier Inc. All rights reserved.

Efficient regeneration of sorghum, Sorghum bicolor (L.) Moench, from shoot-tip explant.

PubMed

Syamala, D; Devi, Prathibha

2003-12-01

Novel protocols for production of multiple shoot-tip clumps and somatic embryos of Sorghum bicolor (L.) Moench were developed with long-term goal of crop improvement through genetic transformation. Multiple shoot-tip clumps were developed in vitro from shoot-tip explant of one-week old seedling, cultured on MS medium containing only BA (0.5, 1 or 2 mg/l) or both BA (1 or 2 mg/l) and 2,4-D (0.5 mg/l) with bi-weekly subculture. Somatic embryos were directly produced on the enlarged dome shaped growing structures that developed from the shoot-tips of one-week old seedling explants (without any callus formation) when cultured on MS medium supplemented with both 2,4-D (0.5 mg/l) and BA (0.5 mg/l). However, the supplementation of MS medium with only 2,4-D (0.5 mg/l) induced compact callus without any plantlet regeneration. Each multiple shoot-clump was capable of regenerating more than 80 shoots via an intensive differentiation of both axillary and adventitious shoot buds, the somatic embryos were capable of 90% germination, plant conversion and regeneration. The regenerated shoots could be efficiently rooted on MS medium containing indole-3-butyric acid (IBA 1 mg/l). The plants were successfully transplanted to glasshouse and grown to maturity with a survival rate of 98%. Morphogenetic response of the explants was found to be genotypically independent.

Daily stress, presleep arousal, and sleep in healthy young women: a daily life computerized sleep diary and actigraphy study.

PubMed

Winzeler, Katja; Voellmin, Annette; Schäfer, Valérie; Meyer, Andrea H; Cajochen, Christian; Wilhelm, Frank H; Bader, Klaus

2014-03-01

Our study aimed to further elucidate the mediating role of presleep arousal in the relationship between daily stress and sleep by investigating subjective sleep quality and actigraphy-assessed sleep efficiency (SE) on both within- and between-participant levels in a sample of healthy young women. Multilevel modeling was applied on electronically assessed data comprising 14 consecutive nights in 145 healthy young women to assess the relationship between daily stress, presleep (somatic and cognitive) arousal, and sleep on both levels between participants and within participants across days. Higher levels of daily stress were consistently and significantly associated with higher levels of somatic and cognitive arousal. Somatic arousal mediated the relationship between daily stress and worsened subjective sleep quality on the between-participant level, while cognitive arousal mediated the relationship between daily stress and worsened subjective sleep quality on the within-participants level. Unexpectedly, healthy young women showed higher SE following days with above-average stress with somatic arousal mediating this relationship. Our data corroborate the role of presleep arousal mediating the relationship between daily stress and subjective sleep quality. Interestingly this effect was restricted to somatic arousal being relevant on interindividual levels and cognitive arousal on intraindividual levels. For young and healthy individuals who experience high stress and arousal, well-established cognitive-behavioral techniques could be useful to regulate arousal and prevent worse subjective sleep quality. Copyright © 2014 Elsevier B.V. All rights reserved.

Effects of interval between fusion and activation, cytochalasin B treatment, and number of transferred embryos, on cloning efficiency in goats.

PubMed

Liu, J; Li, L L; Du, S; Bai, X Y; Zhang, H D; Tang, S; Zhao, M T; Ma, B H; Quan, F S; Zhao, X E; Zhang, Y

2011-10-01

To improve the efficiency of somatic cell nuclear transfer (SCNT) in goats, we evaluated the effects of the interval between fusion and activation (1 to 5 h), cytochalasin B (CB) treatment after electrofusion, and the number of transferred embryos on the in vivo and in vitro development of cloned caprine embryos. The majority of the reconstructed embryos had condensed chromosomes and metaphase-like chromosomes at 2 and 3 h after fusion; cleavage and blastocyst rates from those two groups were higher (P < 0.05) than those of embryos activated 1, 4, or 5 h after fusion. Treatment with CB between fusion and activation improved in vitro and in vivo development of nuclear transfer (NT) goat embryos by reducing the fragmentation rate (P < 0.05). Although there were no significant differences in NT efficiency, pregnancy rate and kids born per recipient were increased by transfer of 20 or 30 embryos per recipient compared with 10 embryos. We concluded that CB treatment for 2 to 3 h between fusion and activation was an efficient method for generating cloned goats by somatic cell NT. In addition, increasing the number of embryos transferred to each recipient resulted in more live offspring from fewer recipients. Copyright © 2011 Elsevier Inc. All rights reserved.

In-depth Proteomics Characterization of Embryogenesis of the Honey Bee Worker (Apis mellifera ligustica) *

PubMed Central

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

2014-01-01

Identifying proteome changes of honey bee embryogenesis is of prime importance for unraveling the molecular mechanisms that they underlie. However, many proteomic changes during the embryonic period are not well characterized. We analyzed the proteomic alterations over the complete time course of honey bee worker embryogenesis at 24, 48, and 72 h of age, using mass spectrometry-based proteomics, label-free quantitation, and bioinformatics. Of the 1460 proteins identified the embryo of all three ages, the core proteome (proteins shared by the embryos of all three ages, accounting for 40%) was mainly involved in protein synthesis, metabolic energy, development, and molecular transporter, which indicates their centrality in driving embryogenesis. However, embryos at different developmental stages have their own specific proteome and pathway signatures to coordinate and modulate developmental events. The young embryos (<24 h) stronger expression of proteins related to nutrition storage and nucleic acid metabolism may correlate with the cell proliferation occurring at this stage. The middle aged embryos (24–48 h) enhanced expression of proteins associated with cell cycle control, transporters, antioxidant activity, and the cytoskeleton suggest their roles to support rudimentary organogenesis. Among these proteins, the biological pathways of aminoacyl-tRNA biosynthesis, β-alanine metabolism, and protein export are intensively activated in the embryos of middle age. The old embryos (48–72 h) elevated expression of proteins implicated in fatty acid metabolism and morphogenesis indicate their functionality for the formation and development of organs and dorsal closure, in which the biological pathways of fatty acid metabolism and RNA transport are highly activated. These findings add novel understanding to the molecular details of honey bee embryogenesis, in which the programmed activation of the proteome matches with the physiological transition observed during embryogenesis. The identified biological pathways and key node proteins allow for further functional analysis and genetic manipulation for both the honey bee embryos and other eusocial insects. PMID:24895377

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

PubMed

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

2014-09-01

Identifying proteome changes of honey bee embryogenesis is of prime importance for unraveling the molecular mechanisms that they underlie. However, many proteomic changes during the embryonic period are not well characterized. We analyzed the proteomic alterations over the complete time course of honey bee worker embryogenesis at 24, 48, and 72 h of age, using mass spectrometry-based proteomics, label-free quantitation, and bioinformatics. Of the 1460 proteins identified the embryo of all three ages, the core proteome (proteins shared by the embryos of all three ages, accounting for 40%) was mainly involved in protein synthesis, metabolic energy, development, and molecular transporter, which indicates their centrality in driving embryogenesis. However, embryos at different developmental stages have their own specific proteome and pathway signatures to coordinate and modulate developmental events. The young embryos (<24 h) stronger expression of proteins related to nutrition storage and nucleic acid metabolism may correlate with the cell proliferation occurring at this stage. The middle aged embryos (24-48 h) enhanced expression of proteins associated with cell cycle control, transporters, antioxidant activity, and the cytoskeleton suggest their roles to support rudimentary organogenesis. Among these proteins, the biological pathways of aminoacyl-tRNA biosynthesis, β-alanine metabolism, and protein export are intensively activated in the embryos of middle age. The old embryos (48-72 h) elevated expression of proteins implicated in fatty acid metabolism and morphogenesis indicate their functionality for the formation and development of organs and dorsal closure, in which the biological pathways of fatty acid metabolism and RNA transport are highly activated. These findings add novel understanding to the molecular details of honey bee embryogenesis, in which the programmed activation of the proteome matches with the physiological transition observed during embryogenesis. The identified biological pathways and key node proteins allow for further functional analysis and genetic manipulation for both the honey bee embryos and other eusocial insects. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Efficient Production of Retroviruses Using PLGA/bPEI-DNA Nanoparticles and Application for Reprogramming Somatic Cells

PubMed Central

Do, Eun Kyoung; Cheon, Hyo Cheon; Heo, Soon Chul; Kwon, Yang Woo; Jeong, Geun Ok; Kim, Ba Reun; Kim, Jae Ho

2013-01-01

Reprogramming of somatic cells to pluripotent cells requires the introduction of factors driving fate switches. Viral delivery has been the most efficient method for generation of induced pluripotent stem cells. Transfection, which precedes virus production, is a commonly-used process for delivery of nucleic acids into cells. The aim of this study is to evaluate the efficiency of PLGA/ bPEI nanoparticles in transfection and virus production. Using a modified method of producing PLGA nanoparticles, PLGA/bPEI-DNA nanoparticles were examined for transfection efficiency and virus production yield in comparison with PLGA-DNA, bPEI-DNA nanoparticles or liposome-DNA complexes. After testing various ratios of PLGA, bPEI, and DNA, the ratio of 6:3:1 (PLGA:bPEI:DNA, w/w/w) was determined to be optimal, with acceptable cellular toxicity. PLGA/bPEI-DNA (6:3:1) nanoparticles showed superior transfection efficiency, especially in multiple gene transfection, and viral yield when compared with liposome-DNA complexes. The culture supernatants of HEK293FT cells transfected with PLGA/bPEI-DNA of viral constructs containing reprogramming factors (Oct4, Sox2, Klf4, or c-Myc) successfully and more efficiently generated induced pluripotent stem cell colonies from mouse embryonic fibroblasts. These results strongly suggest that PLGA/bPEI-DNA nanoparticles can provide significant advantages in studying the effect of multiple factor delivery such as in reprogramming or direct conversion of cell fate. PMID:24098810

The Pesticide Malathion Disrupts "Xenopus" and Zebrafish Embryogenesis: An Investigative Laboratory Exercise in Developmental Toxicology

ERIC Educational Resources Information Center

Chemotti, Diana C.; Davis, Sarah N.; Cook, Leslie W.; Willoughby, Ian R.; Paradise, Christopher J.; Lom, Barbara

2006-01-01

Malathion is an organophosphorus insecticide, which is often sprayed to control mosquitoes. When applied to aquatic habitats, malathion can also influence the embryogenesis of non-target organisms such as frogs and fish. We modified the frog embryo teratogen assay in "Xenopus" (FETAX), a standard toxicological assay, into an investigative…

DNA methyltransferase expressions in Japanese rice fish (Oryzias latipes) embryogenesis is developmentally regulated and modulated by ethanol and 5-azacytidine

USDA-ARS?s Scientific Manuscript database

We aimed to investigate the impact of the epigenome in inducting fetal alcohol spectrum disorder (FASD) phenotypes in Japanese rice fish embryogenesis. One of the significant events in epigenome is DNA methylation which is catalyzed by DNA methyl transferase (DNMT) enzymes. We analyzed DNMT enzyme m...

Developmental regulation of neuroligin genes in Japanese rice fish (oryzias latipes) embryogenesis maintains the rhythym during ethanol-in

USDA-ARS?s Scientific Manuscript database

Although prenatal alcohol exposure is the potential cause of fetal alcohol spectrum disorder (FASD) in humans, the molecular mechanism(s) of FASD is yet unknown. We have used Japanese rice fish (Oryzias latipes) embryogenesis as an animal model of FASD and reported that this model has effectively ge...

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.

Direct Reprogramming of Murine Fibroblasts to Hematopoietic Progenitor Cells

PubMed Central

Batta, Kiran; Florkowska, Magdalena; Kouskoff, Valerie; Lacaud, Georges

2014-01-01

Summary Recent reports have shown that somatic cells, under appropriate culture conditions, could be directly reprogrammed to cardiac, hepatic, or neuronal phenotype by lineage-specific transcription factors. In this study, we demonstrate that both embryonic and adult somatic fibroblasts can be efficiently reprogrammed to clonal multilineage hematopoietic progenitors by the ectopic expression of the transcription factors ERG, GATA2, LMO2, RUNX1c, and SCL. These reprogrammed cells were stably expanded on stromal cells and possessed short-term reconstitution ability in vivo. Loss of p53 function facilitated reprogramming to blood, and p53−/− reprogrammed cells efficiently generated erythroid, megakaryocytic, myeloid, and lymphoid lineages. Genome-wide analyses revealed that generation of hematopoietic progenitors was preceded by the appearance of hemogenic endothelial cells expressing endothelial and hematopoietic genes. Altogether, our findings suggest that direct reprogramming could represent a valid alternative approach to the differentiation of embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) for disease modeling and autologous blood cell therapies. PMID:25466247

Induced Pluripotency and Gene Editing in Disease Modelling: Perspectives and Challenges

PubMed Central

Seah, Yu Fen Samantha; EL Farran, Chadi A.; Warrier, Tushar; Xu, Jian; Loh, Yuin-Han

2015-01-01

Embryonic stem cells (ESCs) are chiefly characterized by their ability to self-renew and to differentiate into any cell type derived from the three main germ layers. It was demonstrated that somatic cells could be reprogrammed to form induced pluripotent stem cells (iPSCs) via various strategies. Gene editing is a technique that can be used to make targeted changes in the genome, and the efficiency of this process has been significantly enhanced by recent advancements. The use of engineered endonucleases, such as homing endonucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Cas9 of the CRISPR system, has significantly enhanced the efficiency of gene editing. The combination of somatic cell reprogramming with gene editing enables us to model human diseases in vitro, in a manner considered superior to animal disease models. In this review, we discuss the various strategies of reprogramming and gene targeting with an emphasis on the current advancements and challenges of using these techniques to model human diseases. PMID:26633382

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

Chromosome microduplication in somatic cells decreases the genetic stability of human reprogrammed somatic cells and results in pluripotent stem cells.

PubMed

Yu, Yang; Chang, Liang; Zhao, Hongcui; Li, Rong; Fan, Yong; Qiao, Jie

2015-05-12

Human pluripotent stem cells, including cloned embryonic and induced pluripotent stem cells, offer a limitless cellular source for regenerative medicine. However, their derivation efficiency is limited, and a large proportion of cells are arrested during reprogramming. In the current study, we explored chromosome microdeletion/duplication in arrested and established reprogrammed cells. Our results show that aneuploidy induced by somatic cell nuclear transfer technology is a key factor in the developmental failure of cloned human embryos and primary colonies from implanted cloned blastocysts and that expression patterns of apoptosis-related genes are dynamically altered. Overall, ~20%-53% of arrested primary colonies in induced plurpotent stem cells displayed aneuploidy, and upregulation of P53 and Bax occurred in all arrested primary colonies. Interestingly, when somatic cells with pre-existing chromosomal mutations were used as donor cells, no cloned blastocysts were obtained, and additional chromosomal mutations were detected in the resulting iPS cells following long-term culture, which was not observed in the two iPS cell lines with normal karyotypes. In conclusion, aneuploidy induced by the reprogramming process restricts the derivation of pluripotent stem cells, and, more importantly, pre-existing chromosomal mutations enhance the risk of genome instability, which limits the clinical utility of these cells.

Cloning from stem cells: different lineages, different species, same story.

PubMed

Oback, Björn

2009-01-01

Following nuclear transfer (NT), the most stringent measure of extensive donor cell reprogramming is development into viable offspring. This is referred to as cloning efficiency and quantified as the proportion of cloned embryos transferred into surrogate mothers that survive into adulthood. Cloning efficiency depends on the ability of the enucleated recipient cell to carry out the reprogramming reactions ('reprogramming ability') and the ability of the nuclear donor cell to be reprogrammed ('reprogrammability'). It has been postulated that reprogrammability of the somatic donor cell epigenome is inversely proportional to its differentiation status. In order to test this hypothesis, reprogrammability was compared between undifferentiated stem cells and their differentiated isogenic progeny. In the mouse, cells of divergent differentiation status from the neuronal, haematopoietic and skin epithelial lineage were tested. In cattle and deer, skeletal muscle and antler cells, respectively, were used as donors. No conclusive correlation between differentiation status and cloning efficiency was found, indicating that somatic donor cell type may not be the limiting factor for cloning success. This may reflect technical limitations of the NT-induced reprogramming assay. Alternatively, differentiation status and reprogrammability may be unrelated, making all cells equally difficult to reprogramme once they have left the ground state of pluripotency.

Genetic engineering including superseding microinjection: new ways to make GM pigs.

PubMed

Galli, Cesare; Perota, Andrea; Brunetti, Dario; Lagutina, Irina; Lazzari, Giovanna; Lucchini, Franco

2010-01-01

Techniques for genetic engineering of swine are providing genetically modified animals of importance for the field of xenotransplantation, animal models for human diseases and for a variety of research applications. Many of these modifications have been directed toward avoiding naturally existing cellular and antibody responses to species-specific antigens. A number of techniques are today available to engineering the genome of mammals, these range from the well established less efficient method of DNA microinjection into the zygote, the use of viral vectors, to the more recent use of somatic cell nuclear transfer. The use of enzymatic engineering that are being developed now will refine the precision of the genetic modification combined with the use of new vectors like transposons. The use of somatic cell nuclear transfer is currently the most efficient way to generate genetically modified pigs. The development of enzymatic engineering with zinc-finger nucleases, recombinases and transposons will revolutionize the field. Nevertheless, genetic engineering in large domesticated animals will remain a challenging task. Recent improvements in several fields of cell and molecular biology offer new promises and opportunities toward an easier, cost-effective and efficient generation of transgenic pigs. © 2010 John Wiley & Sons A/S.

Reprogramming towards totipotency is greatly facilitated by synergistic effects of small molecules

PubMed Central

Tajima, Yosuke; Yoshida, Koki; Oikawa, Mami; Azuma, Rika; Allen, George E.; Tsujikawa, Tomomi; Tsukaguchi, Tomomasa; Bradshaw, Charles R.; Jullien, Jerome; Yamagata, Kazuo; Matsumoto, Kazuya; Anzai, Masayuki; Imai, Hiroshi; Gurdon, John B.; Yamada, Masayasu

2017-01-01

ABSTRACT Animal cloning has been achieved in many species by transplanting differentiated cell nuclei to unfertilized oocytes. However, the low efficiencies of cloning have remained an unresolved issue. Here we find that the combination of two small molecules, trichostatin A (TSA) and vitamin C (VC), under culture condition with bovine serum albumin deionized by ion-exchange resins, dramatically improves the cloning efficiency in mice and 15% of cloned embryos develop to term by means of somatic cell nuclear transfer (SCNT). The improvement was not observed by adding the non-treated, rather than deionized, bovine serum. RNA-seq analyses of SCNT embryos at the two-cell stage revealed that the treatment with TSA and VC resulted in the upregulated expression of previously identified reprogramming-resistant genes. Moreover, the expression of early-embryo-specific retroelements was upregulated by the TSA and VC treatment. The enhanced gene expression was relevant to the VC-mediated reduction of histone H3 lysine 9 methylation in SCNT embryos. Our study thus shows a simply applicable method to greatly improve mouse cloning efficiency, and furthers our understanding of how somatic nuclei acquire totipotency. PMID:28412714

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

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

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.

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

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

USDA-ARS?s Scientific Manuscript database

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

Effect of a 1800 MHz electromagnetic field emitted during embryogenesis on chick development and hatchability.

PubMed

Pawlak, K; Nieckarz, Z; Sechman, A; Wojtysiak, D; Bojarski, B; Tombarkiewicz, B

2018-06-01

The level of artificial electromagnetic field (EMF) has steadily increased with the development of human civilization. The developing chicken embryo has been considered a good model to study the effects of EMF on living organisms. The aim of the study was to determine the effect of a 1800 MHz electromagnetic field during embryogenesis on the frequency of chick embryo malformations, morphometric parameters of the heart and liver and concentration of corticosterone in blood plasma, lipid and glycogen content in the liver of newly hatched chicks. A 1800 MHz EMF was found to shorten the duration of embryogenesis (earlier pipping and hatching of chicks) while having no effect on the quantity and quality of chicks and on increasing the incidence of embryo malformations. Exposure of chick embryos to EMF caused decreases in relative heart weight and right ventricle wall thickness. The pipping and hatching of chicks can be accelerated by stressful impact of EMF, which is confirmed by a significant increase in plasma corticosterone concentrations and decrease in fat and glycogen in the liver of chicks exposed during embryogenesis on the electromagnetic field with a frequency of 1800 MHz. © 2018 Blackwell Verlag GmbH.

Differential Accumulation of Sunflower Tetraubiquitin mRNAs during Zygotic Embryogenesis and Developmental Regulation of Their Heat-Shock Response.

PubMed Central

Almoguera, C.; Coca, M. A.; Jordano, J.

1995-01-01

We have isolated and sequenced Ha UbiS, a cDNA for a dry-seed-stored mRNA that encodes tetraubiquitin. We have observed differential accumulation of tetraubiquitin mRNAs during sunflower (Helianthus annuus L.) zygotic embryogenesis. These mRNAs were up-regulated during late embryogenesis and reached higher prevalence in the dry seed, where they were found to be associated mainly with provascular tissue. UbiS mRNA, as confirmed by Rnase A protection experiments, accumulated also in response to heat shock, but only in leaves and later during postgerminative development. These novel observations demonstrate expression during seed maturation of specific plant polyubiquitin transcripts and developmental regulation of their heat-shock response. Using ubiquitin antibodies we also detected discrete, seed-specific proteins with distinct temporal expression patterns during zygotic embryogenesis. Some of these patterns were concurrent with UbiS mRNA accumulation in seeds. The most abundant ubiquitin-reacting proteins found in mature seeds were small (16-22 kD) and acidic (isoelectric points of 6.1-7.4). Possible functional implications for UbiS expression elicited from these observations are discussed. PMID:12228401

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.

Transfer of immunoglobulins and antibodies in the hen's egg

PubMed Central

Kramer, T. T.; Cho, H. C.

1970-01-01

The presence of immunoglobulins and antibodies were investigated in the fertile hen's egg during embryogenesis. The egg yolk, egg albumin, amniotic and allantoic fluids, chick embryo serum and intestinal contents were examined for the presence of immunoglobulin and level of antibodies. Immunoglobulin G was not detected in fresh egg albumin, but appeared in the albumin from the 4th day of embryogenesis and persisted through the 16th day. The antibody profile of egg albumin during embryogenesis attained two peaks, which were separated by a trough on the 8th day of embryogenesis. The immunoelectrophoretic pattern of albumin IgG was different from that of egg yolk IgG. The IgG of chick embryo serum was of γ2 mobility on the 12th day of incubation and shifted gradually to the full range of γ1 and γ2 mobilities on the 20th day of incubation. Egg-transmitted antibodies appeared on the 12th day of incubation and attained peak values on the 16th day of incubation. Moderate antibody levels were detected in the amniotic and allantoic fluids from the 12th to the 18th days of incubation. ImagesFIG. 1FIG. 2FIG. 4FIG. 5FIG. 7 PMID:4098593

Histone deacetylase inhibitor significantly improved the cloning efficiency of porcine somatic cell nuclear transfer embryos.

PubMed

Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Yao, Chaogang; Zhou, Yang; Zhu, Jianguo; Lai, Liangxue; Ouyang, Hongsheng; Pang, Daxin

2011-12-01

Valproic acid (VPA), a histone deacetylase inbibitor, has been shown to generate inducible pluripotent stem (iPS) cells from mouse and human fibroblasts with a significant higher efficiency. Because successful cloning by somatic cell nuclear transfer (SCNT) undergoes a full reprogramming process in which the epigenetic state of a differentiated donor nuclear is converted into an embryonic totipotent state, we speculated that VPA would be useful in promoting cloning efficiency. Therefore, in the present study, we examined whether VPA can promote the developmental competence of SCNT embryos by improving the reprogramming state of donor nucleus. Here we report that 1 mM VPA for 14 to 16 h following activation significantly increased the rate of blastocyst formation of porcine SCNT embryos constructed from Landrace fetal fibroblast cells compared to the control (31.8 vs. 11.4%). However, we found that the acetylation level of Histone H3 lysine 14 and Histone H4 lysine 5 and expression level of Oct4, Sox2, and Klf4 was not significantly changed between VPA-treated and -untreated groups at the blastocyst stage. The SCNT embryos were transferred to 38 surrogates, and the cloning efficiency in the treated group was significantly improved compared with the control group. Taken together, we have demonstrated that VPA can improve both in vitro and in vivo development competence of porcine SCNT embryos.

Conditioned Pain Modulation in Women with Irritable Bowel Syndrome

PubMed Central

Jarrett, Monica E.; Shulman, Robert J.; Cain, Kevin C.; Deechakawan, Wimon; Smith, Lynne T.; Richebé, Philippe; Eugenio, Margaret; Heitkemper, Margaret M.

2013-01-01

Evidence suggests that patients with irritable bowel syndrome (IBS) are more vigilant to pain-associated stimuli. The aims of this study were to compare women with IBS (n = 20) to healthy control (HC, n = 20) women on pain sensitivity, conditioned pain modulation (CPM) efficiency and salivary cortisol levels before and after the CPM test; and examine the relationship of CPM efficiency with gastrointestinal, somatic pain, and psychological distress symptoms in each group. Women, ages 20–42, gave consent, completed questionnaires and kept a symptom diary for 2 weeks. CPM efficiency was tested with a heat test stimulus and cold water condition stimulus in a laboratory between 8 and 10 a.m. on a follicular phase day. Salivary cortisol samples were collected just before and after the experimental testing. Compared to the HC group, women with IBS reported more days with gastrointestinal and somatic pain/discomfort, psychological distress, fatigue, and feeling stressed. During the CPM baseline testing women with IBS reported greater pain sensitivity compared to the HC group. In the IBS group, CPM efficiency was associated with the pain impact (PROMIS) measure, daily abdominal pain/discomfort, psychological distress, in particular anxiety. There was no group difference in salivary cortisol levels. Overall, women with IBS exhibit an increased sensitivity to thermal stimuli. Impaired CPM was present in a subset of women with IBS. PMID:24463504

Exopolyphosphatases in nuclear and mitochondrial fractions during embryogenesis of the hard tick Rhipicephalus (Boophilus) microplus.

PubMed

Campos, Eldo; Façanha, Arnoldo R; Costa, Evenilton P; da Silva Vaz, Itabajara; Masuda, Aoi; Logullo, Carlos

2008-11-01

The present work evaluated polyphosphate (poly P) metabolism in nuclear and mitochondrial fractions during Rhipicephalus microplus embryogenesis. Nuclear poly P decreased and activity of exopolyphosphatase (PPX - polyphosphate-phosphohydrolases; EC 3.6.1.11) increased after embryo cellularization until the end of embryogenesis. The utilization of mitochondrial poly P content occurred between embryo cellularization and segmentation stages. Increasing amounts of total RNA extracted from eggs progressively enhanced nuclear PPX activity, whereas it exerted no effect on mitochondrial PPX activity. The decline in total poly P content after the 7th day of embryogenesis does not reflect the free P(i) increase and the total poly P chain length decrease after embryo cellularization. The Km(app) utilizing poly P(3), poly P(15) and poly P(65) as substrate was almost the same for the nuclear fraction (around 1muM), while the affinity for substrate in mitochondrial fraction was around 10 times higher for poly P(3) (Km(app) = 0.2muM) than for poly P(15) (Km(app) = 2.8muM) and poly P(65) (Km(app) = 3.6muM). PPX activity was stimulated by a factor of two by Mg2+ and Co2+ in the nuclear fraction and only by Mg2+ in the mitochondrial fraction. Heparin (20microg/mL) inhibited nuclear and mitochondrial PPX activity in about 90 and 95% respectively. Together, these data are consistent with the existence of two different PPX isoforms operating in the nuclei and mitochondria of the hard tick R. microplus with distinct metal dependence, inhibitor and activator sensitivities. The data also shed new light on poly P biochemistry during arthropod embryogenesis, opening new routes for future comparative studies on the physiological roles of different poly P pools distributed over cell compartments.

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

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

Somatic clonal evolution: A selection-centric perspective.

PubMed

Scott, Jacob; Marusyk, Andriy

2017-04-01

It is generally accepted that the initiation and progression of cancers is the result of somatic clonal evolution. Despite many peculiarities, evolution within populations of somatic cells should obey the same Darwinian principles as evolution within natural populations, i.e. variability of heritable phenotypes provides the substrate for context-specific selection forces leading to increased population frequencies of phenotypes, which are better adapted to their environment. Yet, within cancer biology, the more prevalent way to view evolution is as being entirely driven by the accumulation of "driver" mutations. Context-specific selection forces are either ignored, or viewed as constraints from which tumor cells liberate themselves during the course of malignant progression. In this review, we will argue that explicitly focusing on selection forces acting on the populations of neoplastic cells as the driving force of somatic clonal evolution might provide for a more accurate conceptual framework compared to the mutation-centric driver gene paradigm. Whereas little can be done to counteract the "bad luck" of stochastic occurrences of cancer-related mutations, changes in selective pressures and the phenotypic adaptations they induce can, in principle, be exploited to limit the incidence of cancers and to increase the efficiency of existing and future therapies. This article is part of a Special Issue entitled: Evolutionary principles - heterogeneity in cancer?, edited by Dr. Robert A. Gatenby. Copyright © 2017 Elsevier B.V. All rights reserved.

Purification of a novel, nucleoplasmin-like protein from somatic nuclei.

PubMed Central

Cotten, M; Chalkley, R

1987-01-01

We have purified a nucleoplasmin-like protein from the nuclei of somatic Xenopus laevis cells. This protein possesses a number of the distinctive features of nucleoplasmin isolated from oocytes or unfertilized eggs. The protein is recognized by both monoclonal and polyclonal antisera raised against egg nucleoplasmin. The protein has an oligomeric structure, which must be heated in SDS to completely dissociate, is acidic, phosphorylated and efficiently promotes the in vitro formation of chromatin. We have partially characterized this novel protein and because of its resemblance to nucleoplasmin isolated from oocytes or unfertilized eggs we have named this protein nucleoplasmin S. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:3443097

In vitro development of canine somatic cell nuclear transfer embryos in different culture media.

PubMed

Kim, Dong-Hoon; No, Jin-Gu; Choi, Mi-Kyung; Yeom, Dong-Hyeon; Kim, Dong-Kyo; Yang, Byoung-Chul; Yoo, Jae Gyu; Kim, Min Kyu; Kim, Hong-Tea

2015-01-01

The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos.

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.

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.

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.

Nuclear lamins and peripheral nuclear antigens during fertilization and embryogenesis in mice and sea urchins

NASA Technical Reports Server (NTRS)

Schatten, G.; Schatten, H.; Simerly, C.; Maul, G. G.; Chaly, N.

1985-01-01

Nuclear structural changes during fertilization and embryogenesis in mice and sea urchins are traced using four antibodies. The oocytes from virgin female mice, morulae and blastocytes from mated females, and gametes from the sea urchin Lytechnius variegatis are studied using mouse monoclonal antibodies to nuclear lamin A/C, monoclonal antibody to P1, human autoimmune antibodies to lamin A/C, and to lamin B. The mouse fertilization data reveal no lamins on the oocyte; however, lamins are present on the pronuclei, and chromosomes are found on the oocytes and pronuclei. It is detected that on the sea urchin sperm the lamins are reduced to acrosomal and centriolar fossae and peripheral antigens are around the sperm nucleus. The mouse sperm bind lamin antibodies regionally and do not contain antigens. Lamins and antigens are observed on both pronuclei and chromosomes during sea urchin fertilization. Mouse embryogenesis reveals that lamin A/C is not recognized at morula and blastocyst stages; however, lamin B stains are retained. In sea urchin embryogenesis lamin recognition is lost at the blastrula, gastrula, and plutei stages. It is noted that nuclear lamins lost during spermatogenesis are restored at fertilization and peripheral antigens are associated with the surface of chromosomes during meiosis and mitosis and with the periphery of the pronuclei and nuclei during interphase.

Characterization of genomic sequence showing strong association with polyembryony among diverse Citrus species and cultivars, and its synteny with Vitis and Populus.

PubMed

Nakano, Michiharu; Shimada, Takehiko; Endo, Tomoko; Fujii, Hiroshi; Nesumi, Hirohisa; Kita, Masayuki; Ebina, Masumi; Shimizu, Tokurou; Omura, Mitsuo

2012-02-01

Polyembryony, in which multiple somatic nucellar cell-derived embryos develop in addition to the zygotic embryo in a seed, is common in the genus Citrus. Previous genetic studies indicated polyembryony is mainly determined by a single locus, but the underlying molecular mechanism is still unclear. As a step towards identification and characterization of the gene or genes responsible for nucellar embryogenesis in Citrus, haplotype-specific physical maps around the polyembryony locus were constructed. By sequencing three BAC clones aligned on the polyembryony haplotype, a single contiguous draft sequence consisting of 380 kb containing 70 predicted open reading frames (ORFs) was reconstructed. Single nucleotide polymorphism genotypes detected in the sequenced genomic region showed strong association with embryo type in Citrus, indicating a common polyembryony locus is shared among widely diverse Citrus cultivars and species. The arrangement of the predicted ORFs in the characterized genomic region showed high collinearity to the genomic sequence of chromosome 4 of Vitis vinifera and linkage group VI of Populus trichocarpa, suggesting that the syntenic relationship among these species is conserved even though V. vinifera and P. trichocarpa are non-apomictic species. This is the first study to characterize in detail the genomic structure of an apomixis locus determining adventitious embryony. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Nanotopographical Surfaces for Stem Cell Fate Control: Engineering Mechanobiology from the Bottom

PubMed Central

Chen, Weiqiang; Shao, Yue; Li, Xiang; Zhao, Gang; Fu, Jianping

2015-01-01

Summary During embryogenesis and tissue maintenance and repair in an adult organism, a myriad of stem cells are regulated by their surrounding extracellular matrix (ECM) enriched with tissue/organ-specific nanoscale topographical cues to adopt different fates and functions. Attributed to their capability of self-renewal and differentiation into most types of somatic cells, stem cells also hold tremendous promise for regenerative medicine and drug screening. However, a major challenge remains as to achieve fate control of stem cells in vitro with high specificity and yield. Recent exciting advances in nanotechnology and materials science have enabled versatile, robust, and large-scale stem cell engineering in vitro through developments of synthetic nanotopographical surfaces mimicking topological features of stem cell niches. In addition to generating new insights for stem cell biology and embryonic development, this effort opens up unlimited opportunities for innovations in stem cell-based applications. This review is therefore to provide a summary of recent progress along this research direction, with perspectives focusing on emerging methods for generating nanotopographical surfaces and their applications in stem cell research. Furthermore, we provide a review of classical as well as emerging cellular mechano-sensing and -transduction mechanisms underlying stem cell nanotopography sensitivity and also give some hypotheses in regard to how a multitude of signaling events in cellular mechanotransduction may converge and be integrated into core pathways controlling stem cell fate in response to extracellular nanotopography. PMID:25883674

Transient Overexpression of HvSERK2 Improves Barley Resistance to Powdery Mildew.

PubMed

Li, Yingbo; Li, Qingwei; Guo, Guimei; He, Ting; Gao, Runhong; Faheem, Muhammad; Huang, Jianhua; Lu, Ruiju; Liu, Chenghong

2018-04-18

Somatic embryogenesis receptor-like kinases (SERKs) play an essential role in plant response to pathogen infection. Here we identified three SERK genes ( HvSERK1/2/3 ) from barley, and aimed to determine their implication in defense responses to barley powdery mildew ( Bgh ). Although HvSERK1/2/3 share the characteristic domains of the SERK family, only HvSERK2 was significantly induced in barley leaves during Bgh infection. The expression of HvSERK2 was rapidly induced by hydrogen peroxide (H₂O₂) treatment, but not by treatment with salicylic acid (SA), methyl jasmonate (MeJA), ethephon (ETH), or abscisic acid (ABA). Bioinformatics analysis of the cloned HvSERK2 promoter revealed that it contains several elements responsible for defense responses against pathogens. Promoter functional analysis showed that the HvSERK2 promoter was induced by Bgh and H₂O₂. Subcellular localization analysis of HvSERK2 indicated that it is mainly located on the plasma membrane. Transient overexpression of HvSERK2 in epidermal cells of the susceptible barley cultivar Hua 30 reduced the Bgh haustorium index from 58.6% to 43.2%. This study suggests that the HvSERK2 gene plays a positive role in the improvement of barley resistance to powdery mildew, and provides new insight into the function of SERK genes in the biotic stress response of plants.

Abscisic acid regulation of DC8, a carrot embryonic gene. [Daucus carota

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

Hatzopoulos, P.; Fong, F.; Sung, Z.R.

1990-10-01

DC8 encodes a hydrophylic 66 kilodalton protein located in the cytoplasm and cell walls of carrot (Daucus carota) embryo and endosperm. During somatic embryogenesis, the levels of DC8 mRNA and protein begin to increase 5 days after removal of auxin. To study the role of abscisic acid (ABA) in the regulation of DC8 gene, fluridone, 1-methyl-3-phenyl,-5(3-trifluoro-methyl-phenyl)-4(1H)-pyridinone, was used to inhibit the endogenous ABA content of the embryos. Fluridone, 50 micrograms per milliliter, effectively inhibits the accumulation of ABA in globular-tage embryos. Western and Northern analysis show that when fluridone is added to the culture medium DC8 protein and mRNA decreasemore » to very low levels. ABA added to fluridone supplemented culture media restores the DC8 protein and mRNA to control levels. Globular-stage embryos contain 0.9 to 1.4 {times} 10{sup {minus}7} molar ABA while 10{sup {minus}6} molar exogenously supplied ABA is the optimal concentration for restoration of DC8 protein accumulation in fluridone-treated embryos. The mRNA level is increased after 15 minutes of ABA addition and reaches maximal levels by 60 minutes. Evidence is presented that, unlike other ABA-regulated genes, DC8 is not induced in nonembryonic tissues via desiccation nor addition of ABA.« less

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.

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.

Xylose-rich polysaccharides from the primary walls of embryogenic cell line of Pinus caribaea.

PubMed

Mollard, A; Domon, J M; David, H; Joseleau, J P

1997-08-01

Embryogenic cell lines of Pinus caribaea were isolated from somatic embryogenesis from zygotic embryos. Previous studies showed that the proteins and glycoproteins were characteristic of the embryogenic state. In the present work we were seeking typical feature in the polysaccharide from the cell walls of embryogenic calli at nine days of culture. Sequential extraction with water, ammonium oxalate, dimethyl sulfoxide, sodium borohydride and 4.3 M potassium hydroxide revealed that the extracted polysaccharides contained high proportions of arabinose and significant amounts of xylose. Fractionation of the hydrosoluble polymers on DEAE cellulose afforded a xylose-rich fraction (80% xylose, 24% glucose and lower properties of fucose and mannose). Methylation analysis and 13C-NMR spectra showed that the glycan backbone consisted of beta 1 --> 4 linked xylosyl residues Similar study of the fractions extracted respectively with DMSO and 4.3 M KOH showed the presence of polydisperse glycoxylans but excluded the presence of xyloglucan in significant amount. This could be a characteristic feature of embryogenic cells walls of Pinus caribaea or could be typical of cells grown as calluses. In the various fractions obtained from DEAE cellulose chromatography of the alkaline extract the infrequent occurrence of fucoxylans beside an arabinogalactan showed again the unusual nature of the cell wall polymers of this embryogenic lines, which seems to differ greatly from those found in the primary wall of cells from suspension cultures.

Psychological and interactional characteristics of patients with somatoform disorders: Validation of the Somatic Symptoms Experiences Questionnaire (SSEQ) in a clinical psychosomatic population.

PubMed

Herzog, Annabel; Voigt, Katharina; Meyer, Björn; Wollburg, Eileen; Weinmann, Nina; Langs, Gernot; Löwe, Bernd

2015-06-01

The new DSM-5 Somatic Symptom Disorder (SSD) emphasizes the importance of psychological processes related to somatic symptoms in patients with somatoform disorders. To address this, the Somatic Symptoms Experiences Questionnaire (SSEQ), the first self-report scale that assesses a broad range of psychological and interactional characteristics relevant to patients with a somatoform disorder or SSD, was developed. This prospective study was conducted to validate the SSEQ. The 15-item SSEQ was administered along with a battery of self-report questionnaires to psychosomatic inpatients. Patients were assessed with the Structured Clinical Interview for DSM-IV to confirm a somatoform, depressive, or anxiety disorder. Confirmatory factor analyses, tests of internal consistency and tests of validity were performed. Patients (n=262) with a mean age of 43.4 years, 60.3% women, were included in the analyses. The previously observed four-factor model was replicated and internal consistency was good (Cronbach's α=.90). Patients with a somatoform disorder had significantly higher scores on the SSEQ (t=4.24, p<.001) than patients with a depressive/anxiety disorder. Construct validity was shown by high correlations with other instruments measuring related constructs. Hierarchical multiple regression analyses showed that the questionnaire predicted health-related quality of life. Sensitivity to change was shown by significantly higher effect sizes of the SSEQ change scores for improved patients than for patients without improvement. The SSEQ appears to be a reliable, valid, and efficient instrument to assess a broad range of psychological and interactional features related to the experience of somatic symptoms. Copyright © 2015 Elsevier Inc. All rights reserved.

Genome engineering in cattle: recent technological advancements.

PubMed

Wang, Zhongde

2015-02-01

Great strides in technological advancements have been made in the past decade in cattle genome engineering. First, the success of cloning cattle by somatic cell nuclear transfer (SCNT) or chromatin transfer (CT) is a significant advancement that has made obsolete the need for using embryonic stem (ES) cells to conduct cell-mediated genome engineering, whereby site-specific genetic modifications can be conducted in bovine somatic cells via DNA homologous recombination (HR) and whereby genetically engineered cattle can subsequently be produced by animal cloning from the genetically modified cells. With this approach, a chosen bovine genomic locus can be precisely modified in somatic cells, such as to knock out (KO) or knock in (KI) a gene via HR, a gene-targeting strategy that had almost exclusively been used in mouse ES cells. Furthermore, by the creative application of embryonic cloning to rejuvenate somatic cells, cattle genome can be sequentially modified in the same line of somatic cells and complex genetic modifications have been achieved in cattle. Very recently, the development of designer nucleases-such as zinc finger nucleases (ZFNs) and transcription activator-like effector nuclease (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-has enabled highly efficient and more facile genome engineering in cattle. Most notably, by employing such designer nucleases, genomes can be engineered at single-nucleotide precision; this process is now often referred to as genome or gene editing. The above achievements are a drastic departure from the traditional methods of creating genetically modified cattle, where foreign DNAs are randomly integrated into the animal genome, most often along with the integrations of bacterial or viral DNAs. Here, I review the most recent technological developments in cattle genome engineering by highlighting some of the major achievements in creating genetically engineered cattle for agricultural and biomedical applications.

Development of a Traceability System Based on a SNP Array for Large-Scale Production of High-Value White Spruce (Picea glauca)

PubMed Central

Godbout, Julie; Tremblay, Laurence; Levasseur, Caroline; Lavigne, Patricia; Rainville, André; Mackay, John; Bousquet, Jean; Isabel, Nathalie

2017-01-01

Biological material is at the forefront of research programs, as well as application fields such as breeding, aquaculture, and reforestation. While sophisticated techniques are used to produce this material, all too often, there is no strict monitoring during the “production” process to ensure that the specific varieties are the expected ones. Confidence rather than evidence is often applied when the time comes to start a new experiment or to deploy selected varieties in the field. During the last decade, genomics research has led to the development of important resources, which have created opportunities for easily developing tools to assess the conformity of the material along the production chains. In this study, we present a simple methodology that enables the development of a traceability system which, is in fact a by-product of previous genomic projects. The plant production system in white spruce (Picea glauca) is used to illustrate our purpose. In Quebec, one of the favored strategies to produce elite varieties is to use somatic embryogenesis (SE). In order to detect human errors both upstream and downstream of the white spruce production process, this project had two main objectives: (i) to develop methods that make it possible to trace the origin of plants produced, and (ii) to generate a unique genetic fingerprint that could be used to differentiate each embryogenic cell line and ensure its genetic monitoring. Such a system had to rely on a minimum number of low-cost DNA markers and be easy to use by non-specialists. An efficient marker selection process was operationalized by testing different classification methods on simulated datasets. These datasets were generated using in-house bioinformatics tools that simulated crosses involved in the breeding program for which genotypes from hundreds of SNP markers were already available. The rate of misidentification was estimated and various sources of mishandling or contamination were identified. The method can easily be applied to other production systems for which genomic resources are already available. PMID:28791035

Improving Postural Control in the Battement Tendu: One Teacher's Reflections and Somatic Exercises

ERIC Educational Resources Information Center

Batson, Glenna

2010-01-01

The battement tendu is introduced early in dance training, remaining integral to a dancer's vocabulary. Although appearing relatively simple to execute, the tendu aesthetic takes years to master. One reason might be that efficient performance requires complex coordination of postural balance. Known as postural control, this coordination appears in…

Successful cloning of coyotes through interspecies somatic cell nuclear transfer using domestic dog oocytes.

PubMed

Hwang, Insung; Jeong, Yeon Woo; Kim, Joung Joo; Lee, Hyo Jeong; Kang, Mina; Park, Kang Bae; Park, Jung Hwan; Kim, Yeun Wook; Kim, Woo Tae; Shin, Taeyoung; Hyun, Sang Hwan; Jeung, Eui-Bae; Hwang, Woo Suk

2013-01-01

Interspecies somatic cell nuclear transfer (iSCNT) is an emerging assisted reproductive technology (ART) for preserving Nature's diversity. The scarcity of oocytes from some species makes utilisation of readily available oocytes inevitable. In the present study, we describe the successful cloning of coyotes (Canis latrans) through iSCNT using oocytes from domestic dogs (Canis lupus familiaris or dingo). Transfer of 320 interspecies-reconstructed embryos into 22 domestic dog recipients resulted in six pregnancies, from which eight viable offspring were delivered. Fusion rate and cloning efficiency during iSCNT cloning of coyotes were not significantly different from those observed during intraspecies cloning of domestic dogs. Using neonatal fibroblasts as donor cells significantly improved the cloning efficiency compared with cloning using adult fibroblast donor cells (P<0.05). The use of domestic dog oocytes in the cloning of coyotes in the present study holds promise for cloning other endangered species in the Canidae family using similar techniques. However, there are still limitations of the iSCNT technology, as demonstrated by births of morphologically abnormal coyotes and the clones' inheritance of maternal domestic dog mitochondrial DNA.

Creating genetically modified pigs by using nuclear transfer

PubMed Central

Lai, Liangxue; Prather, Randall S

2003-01-01

Nuclear transfer (NT) is a procedure by which genetically identical individuals can be created. The technology of pig somatic NT, including in vitro maturation of oocytes, isolation and treatment of donor cells, artificial activation of reconstructed oocytes, embryo culture and embryo transfer, has been intensively studied in recent years, resulting in birth of cloned pigs in many labs. While it provides an efficient method for producing transgenic pigs, more importantly, it is the only way to produce gene-targeted pigs. So far pig cloning has been successfully used to produce transgenic pigs expressing the green fluorescence protein, expand transgenic pig groups and create gene targeted pigs which are deficient of alpha-1,3-galactosyltransferase. The production of pigs with genetic modification by NT is now in the transition from investigation to practical use. Although the efficiency of somatic cell NT in pig, when measured as development to term as a proportion of oocytes used, is not high, it is anticipated that the ability of making specific modifications to the swine genome will result in this technology having a large impact not only on medicine but also on agriculture. PMID:14613542

Efficient Direct Lineage Reprogramming of Fibroblasts into Induced Cardiomyocytes Using Nanotopographical Cues.

PubMed

Yoo, Junsang; Chang, Yujung; Kim, Hongwon; Baek, Soonbong; Choi, Hwan; Jeong, Gun-Jae; Shin, Jaein; Kim, Hongnam; Kim, Byung-Soo; Kim, Jongpil

2017-03-01

Induced cardiomyocytes (iCMs) generated via direct lineage reprogramming offer a novel therapeutic target for the study and treatment of cardiac diseases. However, the efficiency of iCM generation is significantly low for therapeutic applications. Here, we show an efficient direct conversion of somatic fibroblasts into iCMs using nanotopographic cues. Compared with flat substrates, the direct conversion of fibroblasts into iCMs on nanopatterned substrates resulted in a dramatic increase in the reprogramming efficiency and maturation of iCM phenotypes. Additionally, enhanced reprogramming by substrate nanotopography was due to changes in the activation of focal adhesion kinase and specific histone modifications. Taken together, these results suggest that nanotopographic cues can serve as an efficient stimulant for direct lineage reprogramming into iCMs.

Cracking the egg: virtual embryogenesis of real robots.

PubMed

Cussat-Blanc, Sylvain; Pollack, Jordan

2014-01-01

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

Assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro.

PubMed

Harrison, Sarah Ellys; Sozen, Berna; Christodoulou, Neophytos; Kyprianou, Christos; Zernicka-Goetz, Magdalena

2017-04-14

Mammalian embryogenesis requires intricate interactions between embryonic and extraembryonic tissues to orchestrate and coordinate morphogenesis with changes in developmental potential. Here, we combined mouse embryonic stem cells (ESCs) and extraembryonic trophoblast stem cells (TSCs) in a three-dimensional scaffold to generate structures whose morphogenesis is markedly similar to that of natural embryos. By using genetically modified stem cells and specific inhibitors, we show that embryogenesis of ESC- and TSC-derived embryos-ETS-embryos-depends on cross-talk involving Nodal signaling. When ETS-embryos develop, they spontaneously initiate expression of mesoderm and primordial germ cell markers asymmetrically on the embryonic and extraembryonic border, in response to Wnt and BMP signaling. Our study demonstrates the ability of distinct stem cell types to self-assemble in vitro to generate embryos whose morphogenesis, architecture, and constituent cell types resemble those of natural embryos. Copyright © 2017, American Association for the Advancement of Science.

Laterally confined growth of cells induces nuclear reprogramming in the absence of exogenous biochemical factors.

PubMed

Roy, Bibhas; Venkatachalapathy, Saradha; Ratna, Prasuna; Wang, Yejun; Jokhun, Doorgesh Sharma; Nagarajan, Mallika; Shivashankar, G V

2018-05-22

Cells in tissues undergo transdifferentiation programs when stimulated by specific mechanical and biochemical signals. While seminal studies have demonstrated that exogenous biochemical factors can reprogram somatic cells into pluripotent stem cells, the critical roles played by mechanical signals in such reprogramming process have not been well documented. In this paper, we show that laterally confined growth of fibroblasts on micropatterned substrates induces nuclear reprogramming with high efficiency in the absence of any exogenous reprogramming factors. We provide compelling evidence on the induction of stem cell-like properties using alkaline phosphatase assays and expression of pluripotent markers. Early onset of reprogramming was accompanied with enhanced nuclear dynamics and changes in chromosome intermingling degrees, potentially facilitating rewiring of the genome. Time-lapse analysis of promoter occupancy by immunoprecipitation of H3K9Ac chromatin fragments revealed that epithelial, proliferative, and reprogramming gene promoters were progressively acetylated, while mesenchymal promoters were deacetylated by 10 days. Consistently, RNA sequencing analysis showed a systematic progression from mesenchymal to stem cell transcriptome, highlighting pathways involving mechanisms underlying nuclear reprogramming. We then demonstrated that these mechanically reprogrammed cells could be maintained as stem cells and can be redifferentiated into multiple lineages with high efficiency. Importantly, we also demonstrate the induction of cancer stemness properties in MCF7 cells grown in such laterally confined conditions. Collectively, our results highlight an important generic property of somatic cells that, when grown in laterally confined conditions, acquire stemness. Such mechanical reprogramming of somatic cells demonstrated here has important implications in tissue regeneration and disease models. Copyright © 2018 the Author(s). Published by PNAS.

Traces of embryogenesis are the same in monozygotic and dizygotic twins: not compatible with double ovulation.

PubMed

Boklage, Charles E

2009-06-01

Common knowledge of over a century has it that monozygotic and dizygotic twinning events occur by unrelated mechanisms: monozygotic twinning 'splits' embryos, producing anomalously re-arranged embryogenic asymmetries; dizygotic twinning begins with independent ovulations yielding undisturbed parallel embryogeneses with no expectation of departures from singleton outcomes. The anomalies statistically associated with twin births are due to the re-arranged embryos of the monozygotics. Common knowledge further requires that dizygotic pairs are dichorionic; monochorionicity is exclusive to monozygotic pairs. These are fundamental certainties in the literature of twin biology. Multiple observations contradict those common knowledge understandings. The double ovulation hypothesis of dizygotic twinning is untenable. Girl-boy twins differ subtly from all other humans of either sex, absolutely not representative of all dizygotics. Embryogenesis of dizygotic twins differs from singleton development at least as much as monozygotic embryogenesis does, and in the same ways, and the differences between singletons and twins of both zygosities represent a coherent system of re-arranged embryogenic asymmetries. Dizygotic twinning and monozygotic twinning have the same list of consequences of anomalous embryogenesis. Those include an unignorable fraction of dizygotic pairs that are in fact monochorionic, plus many more sharing co-twins' cells in tissues other than a common chorion. The idea that monozygotic and dizygotic twinning events arise from the same embryogenic mechanism is the only plausible hypothesis that might explain all of the observations.

CNP stoichiometry of a lipid-synthesising zooplankton, Calanus finmarchicus, from winter to spring bloom in a sub-Arctic sound

NASA Astrophysics Data System (ADS)

Aubert, A. B.; Svensen, C.; Hessen, D. O.; Tamelander, T.

2013-02-01

The aim of this study was to investigate the seasonal stoichiometry of the high-latitude lipid-synthesising copepod Calanus finmarchicus and assess how this would affect dietary demands with season, ontogeny and lipid storage. C:N:P ratios in different stages (adults, copepodite V and IV), in eggs and faecal pellets as well as in bulk food (seston) was analysed in a sub-Arctic Norwegian sound (69° 47'N, 19° 19'E) from late February to mid-May 2009. The period covered the phytoplankton bloom and was divided into four sequences of the bloom based on chl a and seston C:chl a ratio variations. The calculation of the somatic elemental C:N and C:P body ratios (without the lipid storage) indicates that nearly homeostatic control in C. finmarchicus is maintained in somatic tissues within stages, while not if the lipid storage pool is included. Nutrient limitation was assessed calculating threshold elemental ratios based on the somatic body ratios and for different sets of assimilation efficiencies, and indicated a predominant C limitation that may reflect demands for lipid storage. The results suggest that stoichiometric composition and demands in such high-latitude, lipid-storing species strongly depend on stage and season, and the large contribution of storage lipids highlights the need for a two-compartment approach for lipid-synthesising species, with different dietary requirements for somatic growth and for lipid storage.

Somatic cell nuclear transfer followed by CRIPSR/CAS9 microinjection results in highly efficient genome editing in cloned pigs

USDA-ARS?s Scientific Manuscript database

The domestic pig is an ideal “dual purpose” animal model for agricultural and biomedical research. With the availability of genome editing tools [e.g. clustered regularly interspersed short palindromic repeat (CRISPR) and associated nuclease Cas9 (CRISPR/Cas9)] it is now possible to perform site-sp...

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.

Somatization: the under-recognized factor in nonspecific eczema. The Hordaland Health Study (HUSK).

PubMed

Klokk, M; Stansfeld, S; Overland, S; Wilhelmsen, I; Gotestam, K G; Steinshamn, S; Mykletun, A

2011-03-01

Psychodermatology has focused primarily on depression and anxiety in eczema. Skin symptoms are listed among many others for the ICD-10 diagnosis of somatization disorder. Somatization (unexplained somatic symptoms) is highly prevalent in the general population, but its association with eczema is yet to be empirically investigated. We therefore explored the association between somatization and eczema by examining the extent of somatization in eczema compared with allergic rhinitis, and by examining if eczema was more strongly associated with somatization than with anxiety and depression. Finally, we aimed to examine the relationship between the site of eczema and somatization for individual somatic symptoms and for somatic symptoms as a whole. For this population-based cross-sectional study we employed data from the Hordaland Health Study (HUSK) with 15,225 participants aged 41-48 years. Information on nonspecific eczema, allergic rhinitis, somatization, anxiety, depression and other covariates was obtained by self-report. The association between nonspecific eczema and somatization was strong and followed a dose-response pattern, as did all somatic symptoms in our index of somatization when analysed separately. The association between nonspecific eczema and somatization was stronger than that between rhinitis and somatization, and also the association between nonspecific eczema and anxiety and depression. In multivariate models, somatization accounted for most of the association between nonspecific eczema and anxiety/depression. In contrast, the association between nonspecific eczema and somatization was robust for adjustment for anxiety/depression. Somatization was strongly associated with nonspecific eczema. This applies to a whole range of somatic symptoms constituting the construct of somatization. There is hardly any mention of somatization in leading dermatological journals, in contrast to anxiety and depression which are frequently reported in eczema. We speculate that this under-recognition of somatization in the dermatological literature may correspond to under-recognition of this factor also in clinical practice. © 2011 The Authors. BJD © 2011 British Association of Dermatologists.

Mechanical Model of Geometric Cell and Topological Algorithm for Cell Dynamics from Single-Cell to Formation of Monolayered Tissues with Pattern

PubMed Central

Kachalo, Sëma; Naveed, Hammad; Cao, Youfang; Zhao, Jieling; Liang, Jie

2015-01-01

Geometric and mechanical properties of individual cells and interactions among neighboring cells are the basis of formation of tissue patterns. Understanding the complex interplay of cells is essential for gaining insight into embryogenesis, tissue development, and other emerging behavior. Here we describe a cell model and an efficient geometric algorithm for studying the dynamic process of tissue formation in 2D (e.g. epithelial tissues). Our approach improves upon previous methods by incorporating properties of individual cells as well as detailed description of the dynamic growth process, with all topological changes accounted for. Cell size, shape, and division plane orientation are modeled realistically. In addition, cell birth, cell growth, cell shrinkage, cell death, cell division, cell collision, and cell rearrangements are now fully accounted for. Different models of cell-cell interactions, such as lateral inhibition during the process of growth, can be studied in detail. Cellular pattern formation for monolayered tissues from arbitrary initial conditions, including that of a single cell, can also be studied in detail. Computational efficiency is achieved through the employment of a special data structure that ensures access to neighboring cells in constant time, without additional space requirement. We have successfully generated tissues consisting of more than 20,000 cells starting from 2 cells within 1 hour. We show that our model can be used to study embryogenesis, tissue fusion, and cell apoptosis. We give detailed study of the classical developmental process of bristle formation on the epidermis of D. melanogaster and the fundamental problem of homeostatic size control in epithelial tissues. Simulation results reveal significant roles of solubility of secreted factors in both the bristle formation and the homeostatic control of tissue size. Our method can be used to study broad problems in monolayered tissue formation. Our software is publicly available. PMID:25974182

Metabolome Profiling of Partial and Fully Reprogrammed Induced Pluripotent Stem Cells.

PubMed

Park, Soon-Jung; Lee, Sang A; Prasain, Nutan; Bae, Daekyeong; Kang, Hyunsu; Ha, Taewon; Kim, Jong Soo; Hong, Ki-Sung; Mantel, Charlie; Moon, Sung-Hwan; Broxmeyer, Hal E; Lee, Man Ryul

2017-05-15

Acquisition of proper metabolomic fate is required to convert somatic cells toward fully reprogrammed pluripotent stem cells. The majority of induced pluripotent stem cells (iPSCs) are partially reprogrammed and have a transcriptome different from that of the pluripotent stem cells. The metabolomic profile and mitochondrial metabolic functions required to achieve full reprogramming of somatic cells to iPSC status have not yet been elucidated. Clarification of the metabolites underlying reprogramming mechanisms should enable further optimization to enhance the efficiency of obtaining fully reprogrammed iPSCs. In this study, we characterized the metabolites of human fully reprogrammed iPSCs, partially reprogrammed iPSCs, and embryonic stem cells (ESCs). Using capillary electrophoresis time-of-flight mass spectrometry-based metabolomics, we found that 89% of analyzed metabolites were similarly expressed in fully reprogrammed iPSCs and human ESCs (hESCs), whereas partially reprogrammed iPSCs shared only 74% similarly expressed metabolites with hESCs. Metabolomic profiling analysis suggested that converting mitochondrial respiration to glycolytic flux is critical for reprogramming of somatic cells into fully reprogrammed iPSCs. This characterization of metabolic reprogramming in iPSCs may enable the development of new reprogramming parameters for enhancing the generation of fully reprogrammed human iPSCs.

[Construction and characterization of liposomal magnetofection system in pig kidney cells].

PubMed

Chen, Wenjie; Cui, Haixin; Zhao, Xiang; Cui, Jinhui; Wang, Yan; Sun, Changjiao

2014-06-01

Magnetic nano gene vector is one of the non-viral gene vectors, modified by functional group to bind cationic transfect reagents. Coupling magnetofection with the universal lipofection we developed a novel somatic cell transfection method as the so-called liposomal magnetofection (LMF). This approach is potential to provide somatic cell cloning with stable genetic cell lines to cultivate transgenic animals. In order to construct such liposomal magnetic gene vectors complexes system, we used nano magnetic gene vector to combine with liposomal cationic transfect reagents by molecular self-assembly. This vectors system successfully carried exogenous gene and then transfected animal somatic cells. Here, we conducted atomic force microscopy (AFM), zeta potential-diameter analysis and other characterization experiments to investegate the size distribution and morphology of magnetic nanoparticles, the way of the vectors to load and concentrate DNA molecules. Our data reveal that, the LMF of Pig Kidney cells exhibited higher transfection efficiency comparing with the transfection mediated by the commercial lipofectamine2000. Moreover, LMF method overcomes the constraint of transient expression mediated by lipofection. Meanwhile, MTT assay showed low cytotoxicity of LMF. Hence, LMF is a feasible, low cytotoxic and effective method of cell transfection.

Sexually mature transgenic American chestnut trees via embryogenic suspension-based transformation.

PubMed

Andrade, Gisele M; Nairn, Campbell J; Le, Huong T; Merkle, Scott A

2009-09-01

The availability of a system for direct transfer of anti-fungal candidate genes into American chestnut (Castanea dentata), devastated by a fungal blight in the last century, would offer an alternative or supplemental approach to conventional breeding for production of chestnut trees resistant to the blight fungus and other pathogens. By taking advantage of the strong ability of embryogenic American chestnut cultures to proliferate in suspension, a high-throughput Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into the tree was established. Proembryogenic masses (PEMs) were co-cultivated with A. tumefaciens strain AGL1 harboring the plasmid pCAMBIA 2301, followed by stringent selection with 50 or 100 mg/l Geneticin. A protocol employing size-fractionation to enrich for small PEMs to use as target material and selection in suspension culture was applied to rapidly produce transgenic events with an average efficiency of four independent transformation events per 50 mg of target tissue and minimal escapes. Mature somatic embryos, representing 18 transgenic events and derived from multiple American chestnut target genotypes, were germinated and over 100 transgenic somatic seedlings were produced and acclimatized to greenhouse conditions. Multiple vigorous transgenic somatic seedlings produced functional staminate flowers within 3 years following regeneration.

DNA methylation at a bovine alpha satellite I repeat CpG site during development following fertilization and somatic cell nuclear transfer.

PubMed

Couldrey, Christine; Wells, David N

2013-01-01

Incomplete epigenetic reprogramming is postulated to contribute to the low developmental success following somatic cell nuclear transfer (SCNT). Here, we describe the epigenetic reprogramming of DNA methylation at an alpha satellite I CpG site (αsatI-5) during development of cattle generated either by artificial insemination (AI) or in vitro fertilization (IVF) and SCNT. Quantitative methylation analysis identified that SCNT donor cells were highly methylated at αsatI-5 and resulting SCNT blastocysts showed significantly more methylation than IVF blastocysts. At implantation, no difference in methylation was observed between SCNT and AI in trophoblast tissue at αsatI-5, however, SCNT embryos were significantly hyper-methylated compared to AI controls at this time point. Following implantation, DNA methylation at αsatI-5 decreased in AI but not SCNT placental tissues. In contrast to placenta, the proportion of methylation at αsatI-5 remained high in adrenal, kidney and muscle tissues during development. Differences in the average proportion of methylation were smaller in somatic tissues than placental tissues but, on average, SCNT somatic tissues were hyper-methylated at αsatI-5. Although sperm from all bulls was less methylated than somatic tissues at αsatI-5, on average this site remained hyper-methylated in sperm from cloned bulls compared with control bulls. This developmental time course confirms that epigenetic reprogramming does occur, at least to some extent, following SCNT. However, the elevated methylation levels observed in SCNT blastocysts and cellular derivatives implies that there is either insufficient time or abundance of appropriate reprogramming factors in oocytes to ensure complete reprogramming. Incomplete reprogramming at this CpG site may be a contributing factor to low SCNT success rates, but more likely represents the tip of the iceberg in terms of incompletely reprogramming. Until protocols ensure the epigenetic signature of a differentiated somatic cell is reset to a state resembling totipotency, the efficiency of SCNT is likely to remain low.

DNA Methylation at a Bovine Alpha Satellite I Repeat CpG Site during Development following Fertilization and Somatic Cell Nuclear Transfer

PubMed Central

Couldrey, Christine; Wells, David N.

2013-01-01

Incomplete epigenetic reprogramming is postulated to contribute to the low developmental success following somatic cell nuclear transfer (SCNT). Here, we describe the epigenetic reprogramming of DNA methylation at an alpha satellite I CpG site (αsatI-5) during development of cattle generated either by artificial insemination (AI) or in vitro fertilization (IVF) and SCNT. Quantitative methylation analysis identified that SCNT donor cells were highly methylated at αsatI-5 and resulting SCNT blastocysts showed significantly more methylation than IVF blastocysts. At implantation, no difference in methylation was observed between SCNT and AI in trophoblast tissue at αsatI-5, however, SCNT embryos were significantly hyper-methylated compared to AI controls at this time point. Following implantation, DNA methylation at αsatI-5 decreased in AI but not SCNT placental tissues. In contrast to placenta, the proportion of methylation at αsatI-5 remained high in adrenal, kidney and muscle tissues during development. Differences in the average proportion of methylation were smaller in somatic tissues than placental tissues but, on average, SCNT somatic tissues were hyper-methylated at αsatI-5. Although sperm from all bulls was less methylated than somatic tissues at αsatI-5, on average this site remained hyper-methylated in sperm from cloned bulls compared with control bulls. This developmental time course confirms that epigenetic reprogramming does occur, at least to some extent, following SCNT. However, the elevated methylation levels observed in SCNT blastocysts and cellular derivatives implies that there is either insufficient time or abundance of appropriate reprogramming factors in oocytes to ensure complete reprogramming. Incomplete reprogramming at this CpG site may be a contributing factor to low SCNT success rates, but more likely represents the tip of the iceberg in terms of incompletely reprogramming. Until protocols ensure the epigenetic signature of a differentiated somatic cell is reset to a state resembling totipotency, the efficiency of SCNT is likely to remain low. PMID:23383311

Efficient and Heritable Gene Targeting in Tilapia by CRISPR/Cas9

PubMed Central

Li, Minghui; Yang, Huihui; Zhao, Jiue; Fang, Lingling; Shi, Hongjuan; Li, Mengru; Sun, Yunlv; Zhang, Xianbo; Jiang, Dongneng; Zhou, Linyan; Wang, Deshou

2014-01-01

Studies of gene function in non-model animals have been limited by the approaches available for eliminating gene function. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated) system has recently become a powerful tool for targeted genome editing. Here, we report the use of the CRISPR/Cas9 system to disrupt selected genes, including nanos2, nanos3, dmrt1, and foxl2, with efficiencies as high as 95%. In addition, mutations in dmrt1 and foxl2 induced by CRISPR/Cas9 were efficiently transmitted through the germline to F1. Obvious phenotypes were observed in the G0 generation after mutation of germ cell or somatic cell-specific genes. For example, loss of Nanos2 and Nanos3 in XY and XX fish resulted in germ cell-deficient gonads as demonstrated by GFP labeling and Vasa staining, respectively, while masculinization of somatic cells in both XY and XX gonads was demonstrated by Dmrt1 and Cyp11b2 immunohistochemistry and by up-regulation of serum androgen levels. Our data demonstrate that targeted, heritable gene editing can be achieved in tilapia, providing a convenient and effective approach for generating loss-of-function mutants. Furthermore, our study shows the utility of the CRISPR/Cas9 system for genetic engineering in non-model species like tilapia and potentially in many other teleost species. PMID:24709635