A major limiting factor for quinoa cultivation as a grain crop on a large scale are virus diseases, in particularly seed borne diseases. Therefore, a somatic embryogenesis protocol is a necessary tool to produce virus free plants. Somatic embryogenesis offers the possibility of mass production of transgenic plants and therefore can be used easily to study the effect on plants
S. Eisa; H. W. Koyro; K. H. Kogel; J. Imani
This review will summarize molecular and genetic ana- lyses aimed at identifying the mechanisms underlying the sequence of events during plant zygotic embryo- genesis. These events are being studied in parallel with the histological and morphological analyses of somatic embryogenesis. The strength and limitations of somatic embryogenesis as a model system will be discussed briefly. The formation of the zygotic
V. L. Dodeman; G. Ducreux; M. Kreis
Moso bamboo [Phyllostachys heterocycla var. pubescens (Mazel ex J. Houz.) Ohwi] is one of the most important forest crops in China and the rest of Asia. Although many sympodial bamboo tissue culture protocols have been established, there is no protocol available for plantlet regeneration as indicated by callus induction for monopodial bamboos, such as Moso bamboo. In the present report, embryogenic callus induction, embryoid development, and germination were established for Moso bamboo from zygotic seed embryos. Callus was initiated from zygotic embryos after 10–20 d culture on MS media supplemented with 4.0 mg/L 2, 4-D and 0.1 mg/L zeatin (ZT). About 50% of the explants produced calli, and nearly 15% of the calli were found to be embryogenic in nature. These embryogenic calli can be subcultured for proliferation in the Murashige and Skoog media (MS) supplemented with 0.5–2.0 mg/L 2, 4-D. These calli were found to have maintained their capacity for regeneration even after one year of subculture. The viable somatic embryoids regenerated in medium containing 5.0–7.0 mg/L ZT. Nearly 5% of the calli were found capable of regenerating into plantlets directly in MS medium containing 5.0–7.0 mg/L ZT. Root growth was more pronounced when the plantlets were transferred to medium containing 2.0 mg/L NAA. After 30 days of subculture, the plantlets were transferred to a greenhouse.
Wu, Xiao-Li; Gu, Xiao-Ping
Moso bamboo [Phyllostachys heterocycla var. pubescens (Mazel ex J. Houz.) Ohwi] is one of the most important forest crops in China and the rest of Asia. Although many sympodial bamboo tissue culture protocols have been established, there is no protocol available for plantlet regeneration as indicated by callus induction for monopodial bamboos, such as Moso bamboo. In the present report, embryogenic callus induction, embryoid development, and germination were established for Moso bamboo from zygotic seed embryos. Callus was initiated from zygotic embryos after 10-20 d culture on MS media supplemented with 4.0 mg/L 2, 4-D and 0.1 mg/L zeatin (ZT). About 50% of the explants produced calli, and nearly 15% of the calli were found to be embryogenic in nature. These embryogenic calli can be subcultured for proliferation in the Murashige and Skoog media (MS) supplemented with 0.5-2.0 mg/L 2, 4-D. These calli were found to have maintained their capacity for regeneration even after one year of subculture. The viable somatic embryoids regenerated in medium containing 5.0-7.0 mg/L ZT. Nearly 5% of the calli were found capable of regenerating into plantlets directly in MS medium containing 5.0-7.0 mg/L ZT. Root growth was more pronounced when the plantlets were transferred to medium containing 2.0 mg/L NAA. After 30 days of subculture, the plantlets were transferred to a greenhouse. PMID:24349159
Yuan, Jin-Ling; Yue, Jin-Jun; Wu, Xiao-Li; Gu, Xiao-Ping
The effect of phenolic compounds on somatic embryogenesis in Feijoa sellowiana was analysed. The results showed that caffeic acid (140-560 microM) significantly increased somatic embryogenesis induction compared with the control. The presence of phloridzin, even at lower concentrations (11.5 microM), or caffeic acid or phloroglucinol at concentrations greater than 140.0 and 197.5 microM, respectively, inhibited somatic embryo development beyond the globular stage. When somatic embryos were transferred to the germination medium, the highest rates of germination (81.9%) were obtained with embryos induced in the presence of phloroglucinol (79.0 microM). At all concentrations tested, somatic embryos induced in medium containing phloroglucinol germinated at higher rates than those induced in the presence of caffeic acid. Histological and ultrastructural studies showed that somatic embryos were formed in close association with phenolic-rich cells which, in more advanced stages of development, formed a zone isolating the embryo from the maternal tissue. A comparative analysis of total phenolic content indicated that phenolics reached a peak by the third week of culture, independently of the medium used. However, after that period, the amount of phenolic compounds was significantly higher in explants cultured in the presence of phloroglucinol than in those cultured in the control or in caffeic acid-containing medium. Attempts to identify the type of phenolic compounds showed that flavan-3-ols and gallic acid derivatives were mainly produced in phloroglucinol-containing medium, whereas flavanones and dihydroflavonols were also present in medium containing caffeic acid. Flavones were the main phenols detected in the control. The ways in which phenolic compounds may affect somatic embryogenesis are discussed. PMID:18767216
Reis, E; Batista, M T; Canhoto, J M
The induction of somatic embryo development in cell cultures of alfalfa (Medicago sativa), celery (Apium graveolens), and lettuce (Lactuca sativa) was compared for 2,4-dichlorophenoxy-acetic acid (2,4-D) and various phenoxy acid growth regulators. Tests using a series of straight chain extensions to the phenoxy acid side chain indicate that phenoxybutanoic acid is active, whereas the phenoxypropanoic and phenoxypentanoic analogs are inactive for the induction of alfalfa embryogenesis. Side branching on the carbon adjacent to the phenoxy group results in optically active compounds. Racemic mixtures and the (+) enantiomers of the compounds are active for alfalfa embryo induction, whereas the (?) enantiomers are inactive and apparently do not inhibit embryogenesis in any way. Development of alfalfa embryos, as measured by plantlet formation from individual embryos, is improved by 4-(2,4-dichlorophenoxy)butanoic acid and with side branching at the carbon adjacent to the phenoxy group compared with induction with 2,4-D. Similarly, substituted phenoxy acids also enhance somatic embryo development in celery and lettuce when compared with 2,4-D. These results are discussed with reference to earlier studies on the structure activity of various synthetic auxins during cell elongation and with reference to the possible importance of auxin metabolism on subsequent somatic embryo development.
Stuart, David A.; McCall, Carol M.
Somatic embryogenesis is a useful tool of plant breeding. In this context, a procedure for inducing somatic embryogenesis in Prunus incisa leaf explants had been previously developed. The original in vitro protocol relies on picloram treatments and exposure to darkness as inductive conditions, the best frequency of embryogenesis being obtained on the second leaf (F(2)) exposed to 4 ?M picloram during 30 days. The morphological and biochemical changes observed during somatic embryogenesis occur in response to alterations in gene expression regulation patterns. A molecular study was conducted in order to provide deeper insight into the fundamental biological factors involved in the induction of this process using a gene candidate strategy and semi-quantitative reverse transcription polymerase chain reaction analysis. So far, no sequence data related to somatic embryogenesis has been available in cherry. In the present study, we cloned and sequenced cDNA fragments of putative genes encoding auxin-binding protein, cell cycle regulator and somatic embryogenesis receptor kinase. Time-course differential transcript accumulations were observed for all investigated genes in leaves or derived callus tissues during the observation period (first month of culture). Their possible involvement in the sequential steps of the embryogenic pathway (dedifferentiation, cell proliferation, differentiation through somatic embryogenesis) is presented and discussed. PMID:23086274
Ben Mahmoud, Kaouther; Delporte, Fabienne; Muhovski, Yordan; Elloumi, Nadhra; Jemmali, Ahmed; Druart, Philippe
Plant hormones play critical roles in the establishment of somatic embryogenesis. During this\\u000a process, somatic plant cells reverse their state of differentiation, acquire pluripotentiality and\\u000a set up a new developmental program. The identification of the regulatory mechanisms that govern\\u000a the key events of somatic embryogenesis requires molecular and genetic investigations. One critical\\u000a issue is how plant hormones and growth regulators act
Clément Thomas; Víctor Jiménez
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
Ayil-Gutiérrez, Benajmín; Galaz-Ávalos, Rosa; Peña-Cabrera, Eduardo; Loyola-Vargas, Victor
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.
Ayil-Gutierrez, Benajmin; Galaz-Avalos, Rosa Maria; Pena-Cabrera, Eduardo; Loyola-Vargas, Victor Manuel
Somatic embryogenesis is the developmental process by which somatic cells undergo restructuring to generate embryogenic cells. These cells then go through a series of morphological and biochemical changes that result in the formation of a somatic or non-zygotic embryo capable of regenerating plants. Somatic embryogenesis represents a unique developmental pathway that includes a number of characteristic events: dedifferentiation of cells,
Xiyan Yang; Xianlong Zhang
Medicago truncatula is a model legume, whose genome is currently being sequenced. Somatic embryogenesis (SE) is a genotype-dependent character and not yet fully understood. In this study, a proteomic approach was used to compare the induction and expression phases of SE of both the highly embryogenic line M9-10a of M. truncatula cv. Jemalong and its non-embryogenic predecessor line, M9. The statistical analysis between the lines revealed 136 proteins with significant differential expression (P < 0.05). Of these, 5 had a presence/absence pattern in M9 vs M9-10a and 22 showed an at least twofold difference in terms of spot volume, were considered of particular relevance to the SE process and therefore chosen for identification. Spots were excised in gel digested with trypsin and proteins were identified using matrix-assisted laser desorption ionization-time of flight/time of flight. Identified proteins indicated a higher adaptability of the embryogenic line toward the stress imposed by the inducing culture conditions. Also, some proteins were shown to have a dual pattern of expression: peroxidase, pyrophosphatase and aspartate aminotransferase. These proteins showed higher expression during the induction phases of the M9 line, whereas in the embryogenic line had higher expression at stages coinciding with embryo formation. PMID:22497501
Almeida, André M; Parreira, José R; Santos, Romana; Duque, Ana Sofia; Francisco, Rita; Tomé, Daniel F A; Ricardo, Cândido Pinto; Coelho, Ana Varela; Fevereiro, Pedro
Secondary somatic embryogenesis is the phenomenon whereby new somatic embryos are initiated from somatic embryos. Such cultures have been described in at least 80 Gymnosperm and Angiosperm species. In the initial step (primary somatic embryogenesis) such cultures have to be started from plant explants. In general, primary somatic embryogenesis from vegetative plant explants is, indirect and mostly driven by auxin
C. J. J. M. Raemakers; E. Jacobsen; R. G. F. Visser
Guava (Psidium guajava L.) is well known for edible fruit, environment friendly pharmaceutical and commercial products for both national and international market. The conventional propagation and in vitro organogenesis do not meet the demand for the good quality planting materials. Somatic embryogenesis for efficient micropropagation of guava (P. guajava L.) has been developed to fill up the gap. Somatic embryogenesis and plantlets regeneration are achieved from 10-week post-anthesis zygotic embryo explants by 8-day inductive treatment with different concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D) on MS agar medium containing 5% sucrose. Subsequent development and maturation of somatic embryos occur after 8 days on MS basal medium supplemented with 5% sucrose without plant growth regulator. The process of somatic embryogenesis shows the highest relative efficiency in 8-day treatment of zygotic embryo explants with 1.0 mg L(-1) 2,4-D. High efficiency germination of somatic embryos and plantlet regeneration takes place on half strength semisolid MS medium amended with 3% sucrose within 2 weeks of subculture. Somatic plantlets are grown for additional 2 weeks by subculturing in MS liquid growth medium containing 3% sucrose. Well-grown plantlets from liquid medium have survived very well following 2-4 week hardening process. The protocol of somatic embryogenesis is optimized for high efficiency micropropagation of guava species. PMID:23179697
The present invention relates to the field of somatic embryo production, particularly to methods for somatic embyrogenesis of Jatropha from ovules. More specifically, the present invention relates to a method and media compositions for somatic embryogenesis of Jatropha curcas from ovules of unopened flower buds. The method is well suited for Jatropha curcas transformation, for producing clonal planting stock useful for large scale Jatropha curcas plantation and for producing haploids, double haploids, diploids and disease-free plantlets.
Factors affecting somatic embryogenesis from root explants of Prunus incisa Thunb. cv. February Pink were investigated. Using a medium containing Murashige and Skoog salts and vitamins supplemented with 10 microM 2,4-dichlorophenoxyacetic (2,4-D), we evaluated the effects of light, growth regulators, amino acids, carbohydrate source, and root induction medium. Explants cultured under light or dark conditions both resulted in the formation of embryos. Embryogenesis was inhibited by the addition of 6-benzyladenine, thidiazuron, or gibberellic acid to the medium. Amino acids were not effective in promoting embryogenesis, with high levels of amino acids actually inhibiting it. Sucrose and glucose effectively induced embryogenesis, while sorbitol and mannitol completely inhibited it. Sucrose and glucose also promoted secondary embryogenesis. Embryos that formed in medium containing 4% or 5% sucrose were abnormally shaped and did not fully develop, while those that formed in medium with sucrose concentrations of 2% or 3% were much more vigorous. Root explants that were induced on medium containing 1.0 micro M indole-3-butyric acid (IBA) produced more somatic embryos than explants induced on medium without IBA. Approximately 50% of the roots induced on medium containing 1.0 microM IBA produced somatic embryos on medium containing 10 microM 2,4-D and 3% sucrose. PMID:15022015
Cheong, E J; Pooler, M R
A highly reproducible system for efficient somatic embryogenesis was developed to regenerate plantlets from cotton (Gossypium hirsutum L.) cultivars (Nazilli M-503 and Nazilli 143). Shoot apices, hypocotyls and nodes of 10-d-old seedlings were used as explants. High frequency (100 %) embryogenic calli was initiated from all tested explants on Murashige and Skoog (1962) (MS) media supplemented with 1 g dm-3
Y. Aydin; Z. Ipekci; T. Talas-O?ra?; A. Zehir; K. Bajrovic; N. Gozukirmizi
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.
Steinmacher, D. A.; Krohn, N. G.; Dantas, A. C. M.; Stefenon, V. M.; Clement, C. R.; Guerra, M. P.
Somatic embryogenesis and whole plant regeneration were achieved in callus cultures derived from hypocotyl, cotyledon and leaf explants excised from seedlings of Gymnema sylvestre. Embryogenic callus was induced on Murashige and Skoog (MS) medium containing 2,4-D (0.5–5.0 µM) +BA (0.5–2.0 µM) and 2% (w\\/v) sucrose in 6–8 weeks of culture. Globular\\/heart stage embryos developed on induction medium. These embryos produced
H. G. Ashok Kumar; H. N. Murthy; K. Y. Paek
Brassinolide (BR), which is the most biologically active brassinosteroid, was used to examine the potential effect of hormone\\u000a on cotton somatic embryogenesis. Ten-day-old cotton (Gossypium hirsutum L., cv. Cooker) seedlings were used for explant source and hypocotyls were removed and cultured on MS basal medium with B5 vitamins supplemented with 1 mg\\/L 6-benzylaminopurine + 0.5 mg\\/L kinetin for callus induction.
Y?ld?z Aydin; Tijen Talas-Ogras; Zeliha Ipekçi-Altas; Nermin Gözükirmizi
In the present study, the procedures for induction of somatic embryogenesis (SE) in an in vitro culture of the tulip have been developed. SE was initiated on flower stem explants isolated from “Apeldoorn” bulbs during\\u000a their low-temperature treatment. Bulbs had not been chilled or had been chilled for 12 or 24 weeks at 5C. The explants were\\u000a cultured with exogenous auxins
Agata Ptak; Anna Bach
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. PMID:18280985
El Abidine Triqui, Zine; Guédira, Abdelkarim; Chlyah, Averil; Chlyah, Hassane; Souvannavong, Vongthip; Haïcour, Robert; Sihachakr, Darasinh
Morphohistological analysis and histochemical studies were carried out during the induction and development of Feijoa sellowiana somatic embryos. Zygotic embryos were cultured on LPm medium containing 2,4-dichlorophenoxyacetic acid (20 microM) and glutamine (8 mM). Somatic embryogenesis could be induced from embryogenic cells that originated in meristematic centers or from clusters of cells. The presence of few starch grains and abundant protein bodies was observed in the globular and early torpedo stages, while in torpedo and cotyledonary-stage somatic embryos an enhanced synthesis of starch grains was associated with the accumulation of reserves to be used in the conversion of the embryos to plantlets. Proteins were predominantly observed in protoderm cells, as well as in the meristematic apical region of torpedo and cotyledonary-stage somatic embryos. PMID:15726807
Cangahuala-Inocente, G C; Steiner, N; Santos, M; Guerra, M P
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.
Bennett, J. Rasheed; Prakash, C. S.
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.
Bennett, J. Rasheed; Prakash, C. S.
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. PMID:23987809
Hill, Robert D; Huang, Shuanglong; Stasolla, Claudio
Somatic embryogenesis was achieved in four leguminous tree species, that is, Acacia catechu, Acacia arabica, Hardwickia binata, and Dalbergia sissoo using immature zygotic embryos as explants on Murashige and Skoog (MS) medium supplemented with 0.25–1.0?mg/l Kn (kinetin) and 2.0–3.0?mg/l 2,4-D (2,4-dichlorophenoxyacetic acid) or NAA (1-napthaleneacetic acid) and 3% sucrose. MS medium containing 2.0?mg/l 2,4-D and 1.0–1.5?mg/l Kn was noted to be most effective in inducing friable embryogenic callus (FEC); the number of somatic embryos per culture varied in MS medium supplemented with 1.0–2.0?mg/l 2,4-D or NAA and 0.25–1.5?mg/l kinetin. The maximum number of somatic embryos was obtained in MS medium containing 1.5–2.0?mg/l 2,4-D or NAA and 1.0–1.5?mg/l kinetin; proliferation of embryogenic calli was enhanced in cultures having 1.0–2.0?mg/l 2,4-D, 1.0–1.5?mg/l kinetin, and 400–600?mg/l L-Proline. The somatic embryos in various shapes and sizes after the first subculture on MS medium supplemented with 0.1?mg/l IAA and 0.25?mg/l BA; developed shoots and rooted in 1/2 strength MS medium supplemented with 0.1?mg/l IBA or IAA. The somatic embryo-derived plantlets were transferred to the field after being hardened in the climate-controlled hardening chamber.
We have developed a reproducible system for efficient direct somatic embryogenesis from leaf and internodal explants of Paulownia elongata. The somatic embryos obtained were subsequently encapsulated as single embryos to produce synthetic seeds. Several plant growth regulators [6-benzylaminopurine, indole-3-acetic acid, alpha-naphthaleneacetic acid, kinetin and thidiazuron (TDZ)] alone or in combination were tested for their capacity to induce somatic embryogenesis. The highest induction frequencies of somatic embryos were obtained on Murashige and Skoog (MS) medium supplemented with 3% sucrose, 0.6% Phytagel, 500 mg l(-1) casein hydrolysate and 10 mg l(-1) TDZ (medium MS10). Somatic embryos were induced from leaf (69.8%) and internode (58.5%) explants on MS10 medium after 7 days. Subsequent withdrawal of TDZ from the induction medium resulted in the maturation and growth of the embryos into plantlets on MS basal media. The maturation frequency of somatic embryos from leaf and internodal explants was 50.8% and 45.8%, respectively. Subculturing of mature embryos led to their germination on the same medium with a germination frequency of 50.1% and 29.8% from leaf and internode explants, respectively. Somatic embryos obtained directly on leaf explants were used for encapsulation in liquid MS medium containing different concentrations of sodium alginate with a 30-min exposure to 50 m M CaCl(2). A 3% sodium alginate concentration provided a uniform encapsulation of the embryos with survival and germination frequencies of 73.7% and 53.3%, respectively. Storage at 4 degrees C for 30 days or 60 days significantly reduced the survival and complete germination frequencies of both encapsulated and non-encapsulated embryos relative to those of non-stored somatic embryos. However, the survival and germination rates of encapsulated embryos increased following storage at 4 degrees C. After 30 days or 60 days of storage, the survival rates of encapsulated embryos were 67.8% and 53.5% and the germination frequencies were 43.2% and 32.4%, respectively. These systems could be useful for the rapid clonal propagation and dissemination of synthetic seed material of Paulownia elongata. PMID:12827435
Ipekci, Z; Gozukirmizi, N
Four critical stages of embryogenesis, including callus induction, cellular acquisition of morphogenetic competence, expression of embryogenic program, and development and maturation of somatic embryos during somatic embryogenesis from leaf discs of eggplant (Solanum melongena L.), were identified by scanning electron microscopy. Temporal changes in arginine decarboxylase (ADC) activity and polyamines (PAs) during critical stages of embryogenesis revealed that high levels of PAs (especially putrescine [PUT]), due to higher ADC activity in discs from the apical region (with high embryogenic capacity) than from the basal region of the leaf (with poor embryogenic capacity), were correlated with differential embryogenesis response. Kinetic studies of the up- and down-regulation of embryogenesis revealed that PUT and difluoromethylarginine pretreatments were most effective before the onset of embryogenesis. Basal discs pretreated with PUT for 4 to 7 d showed improved embryogenesis that was comparable to apical discs. PA content at various critical steps in embryogenesis from basal discs were found to be comparable to that of apical discs following adjustments of cellular PA content by PUT. In contrast, pretreatment of apical discs with difluoromethylarginine for 3 d significantly reduced ADC activity, cellular PA content, and embryogenesis to levels that were comparable to basal discs. Discs from the basal region of leaves treated with PUT for 3 d during the identified stages of embryogenesis improved their embryogenic potential.
Singh Yadav, Jitender; Venkat Rajam, Manchikatla
Carrot somatic embryogenesis was serially observed using a cell cluster immobilizing system with Phytagel. Embryogenic cell\\u000a clusters ranging in size from 32 to 63 ?m were collected by filtration and used for somatic embryo induction. Of the 432 cell\\u000a clusters, 253 grew, i.e., the size of these cell clusters increased, and 192 developed into globular embryos. Through serial\\u000a observation, the
Y. Ibaraki; R. Matsushima; K. Kurata
Summary A regeneration system based on repetitive somatic embryogenesis was developed for peanut (Arachis hypogaea L.). Embryogenic suspension cultures were initiated using individual somatic embryos induced from immature cotyledons cultured on a modified Murashige and Skoog medium containing 40 mg\\/l 2,4-D for 30 days. After transfer to a modified MS liquid medium, the somatic embryos produced masses of secondary and tertiary
Richard E. Durham; Wayne A. Parrott
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.
Steinmacher, D. A.; Guerra, M. P.; Saare-Surminski, K.; Lieberei, R.
Oplopanax elatus is a medicinal plant on the verge of extinction because of overexploitation. In the present study, the effects of various factors on enhancing somatic embryogenesis and plantlet conversion were studied. Mature seeds were collected from a total of 13 plants from 4 mountains in South Korea, and the genetic distances were calculated to analyze the effect of genotype on somatic embryogenesis. Results of cluster analysis and the unweighted-pair-group method with arithmetic mean of 13 genotypes indicated the presence of 3 main groups. Both genotype and explant type affected the induction of somatic embryos (SEs). Sorak 2 and root were found to be the most suitable genotype and explant type, respectively, for SE induction in O. elatus. Among the different types of carbon sources tested, 5% sucrose induced the maximum number of SEs. The formation and development of SEs were significantly influenced by culture density; thus, 10 mg embryonic callus was found to be the most suitable for SE induction. The highest rates of germination and SE conversion were obtained in a germination medium containing 1.8 gelrite and 3.2 g·l(-1) agar. In addition, 80% of the plantlets that were transplanted into artificial soil acclimatized successfully. Thus, our results showed that the percentage survival of O. elatus during in vitro proliferation could be increased by optimizing to the somatic embryogenesis system. PMID:24024109
Moon, Heung-Kyu; Kim, Yong-Wook; Hong, Yong-Pyo; Park, So-Young
Experiments were carried out to investigate the effects of various concentrations of Picloram (0, 1, 2, 3, 6 and 9 mg L(-1)), TDZ (0, 0.5, 1, 1.5 and 2 mg L(-1)), NAA (1.5 mg L(-1)) in combination with TDZ (0.08, 0.2 and 0.4 mg L(-1)), 2,4-D (2.5, 5 and 10 mg L(-1)) combined with BAP (0.25 mg L(-1)) and different types of explants (basal, central and distal part of the bulb scale) on direct somatic embryogenesis induction of Lilium longiflorum var. Ceb-Dazzle. The explants were surface sterilized and cultured on MS medium supplemented with 3% sucrose, 0.3% Phytagel and various concentrations of mentioned growth regulators. It was found that Picloram at a concentration of 2 mg L-' was the most effective treatment for induction of direct somatic embryogenesis and gave the highest number of embryos (18.6) on each explant. The explants from basal part of the bulb scale showed the best responses (19.9 embryos/explant). TDZ alone or combined with NAA in various concentrations was not able to induce somatic embryogenesis, but gave direct bulblet regeneration. Similar results were obtained for 2, 4-D and BAP combination treatments. Induced somatic embryos were transferred to MS medium without growth regulators for maturation and matured plantlets were successfully acclimatized and transferred to in vivo conditions. PMID:19070125
Khosravi, Solmaz; Azghandi, Ali Vatanpour; Mojtahedi, Narges; Haddad, Raheem
Majority of the Indian soybean cultivars are recalcitrant to tissue culture regeneration. The present communication reports the development of somatic embryogenesis in a liquid culture medium from immature cotyledons of G. max. Following induction with 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthalene acetic acid (NAA), the number of somatic embryos and percentage of explants that responded were higher with 45.24 microM 2,4-D. The proliferation of somatic embryos for three successive cycles was achieved in 22.62 microM 2,4-D. Histodifferentiation of somatic embryos under NAA (10.74 microM) indicated that better embryo development and maturation was achieved without any growth regulator. The amino acids such as L-glutamine favoured the somatic embryo induction and histodifferentiation at 20 and 30 mM respectively, where as L-asparagine at 10 mM concentration enhanced the somatic embryo proliferation. In addition, somatic embryos that were desiccated (air-drying method) for 5 days showed better germination (40.88%). The Indian soybean cultivars also showed strict genotypic influence and cv. Pusa 16 was emerged as a best responding cultivar for somatic embryo induction with 74.42% of response. PMID:24266110
Mariashibu, Thankaraj Salammal; Subramanyam, Kondeti; Arun, Muthukrishnan; Theboral, Jeevaraj; Rajesh, Manoharan; Rengan, Sampath Kasthuri; Chakravarthy, Rajan; Manickavasagam, Markandan; Ganapathi, Andy
Somatic embryos were initiated with mature seeds of neem (Azadirachta indica A. Juss.) when cultured on Murashige and Skoog's medium supplemented with thidiazuron (TDZ). Regeneration occurred via somatic\\u000a embryogenesis: direct embryo formation and through an intermediary callus phase. TDZ was very effective and induced somatic\\u000a embryogenesis across a wide range of concentrations (1–50 µm). However, somatic embryogenesis was accompanied by
B. N. S. Murthy; Praveen K. Saxena
Plant regeneration from totipotent cultured cells or protoplasts is a prerequisite for the often proposed genetic modification of plants through somatic cell genetics, and has been achieved in many species. The cereals (and the rest of the Gramineae) have, however, proved to be extremely unresponsive to in vitro culture techniques1. The most convenient source of plant protoplasts is the leaf
Wolfgang Wernicke; Richard Brettell
Embryogenic callus formation in different larch species from Siberia (Larix sibirica, L. gmelinii, and L. sukaczewii) was carried out on MSGm medium supplemented with growth regulators (2.4-D and BAP) and followed one and the same scheme: elongation of somatic cells and their asymmetric division with formation of initial and tube cells. The cells of embryo initial underwent sequential divisions and formed embryonic globules which caused the formation of somatic embryos. Somatic embryos became mature and germinated by addition of ABA and PEG into the medium. Long-term proliferating cell lines and regenerant plants were obtained in Sukachev larch and its hybrid with Siberian larch. The success of somatic embryogenesis depended on the genotype of the donor tree. PMID:23401960
Tret'iakova, I N; Barsukova, A V
Proton induced X-ray emission was used to study the trace elemental profiles of embryogenic and non-embryogenic callus of an important cash crop of India - Plantago ovata. Somatic embryogenesis, a well-known process for plant regeneration and crop improvement is modulated by various factors such as ionizing radiation and micro nutrients in the growth media. The present work reports the trace element variation in normal and irradiated callus tissue of P. ovata. Embryogenic and non-embryogenic callus tissues were exposed to gamma rays from a 60Co gamma source. The absorbed dose ranged from 10 to 100 Gy. Subsequent experiments showed significant dose dependent alterations in K, Ca, Mn, Fe, Ni, Cu, Zn, Br, Sr in both the embryogenic and non-embryogenic callus. The precise involvement of these elements has been discussed in light of somatic embryogenesis of the selected medicinal plant.
Saha, P.; Raychaudhuri, S.; Mishra, D.; Chakraborty, A.; Sudarshan, M.
Somatic embryos were induced directly from immature cotyledons of the genotype of chickpea ICC 4918 (annigiri) on B5 medium supplemented with 2,4,5-T or 2,4-D in combination with BA or KN. Successful transformation was achieved via somatic embryogenesis using Agrobacterium tumefaciens strain LBA4404, carrying a binary plasmid vector system containing neomycin phosphotransferase (NPT II) gene as the selectable marker and beta-glucuronidase (GUS) as a reporter gene. Histochemical staining for GUS expression was observed as primary evidence for transformation. PMID:8979515
Ramana, R V; Venu, C; Jayasree, T; Sadanadam, A
Somatic embryogenesis fromTheobroma cacao L. flower buds, as previously reported on five Forastero hybrid genotypes, was tested on several other genotypes, belonging to the three cocoa-tree groups: Forastero, Trinitario and Criollo. The results gave evidence of genotypic efficiencies. Explants were cultivated under two successive conditions: callogenesis and expression media. The morphological and histological responses were different for embryogenic or non-embryogenic
L. Alemanno; M. Berthouly; N. Michaux-Ferrière
To determine some physiological parameters implicated in somatic embryogenesis in date palm (Phoenix dactylifera L.), peroxidases have been studied. Activated charcoal commonly used in date palm tissue culture as an essential antibrowning\\u000a factor decreased cellular protein contents and peroxidase activities. During the first months of culture, the conventionally\\u000a used medium (100 mg dm?3 of 2,4-dichlorophenoxyacetic acid, 3 g dm?3 charcoal)
I. El Hadrami; M. Baaziz
Summary Somatic embryogenesis from nucellus-derived callus cultures of five cultivars, including three (Caipira, Seleta Vermelha,\\u000a and Valencia) of sweet oranges (C. sinesis L. Osbeck), Rangpur lime (C. limonia L. Osbeck), and Cleopatra mandarin (C. reticulata Blanco) (lines I and II), were studied. Callus lines maintained on MT medium supplemented with 50 g l?1 sucrose were transferred to MT medium supplemented with
Márcio L. Tomaz; Beatriz M. Januzzi Mendes; francisco De Assis A. Mourão Filho; Clarice G. B. Demétrio; Naratip Jansakul; Adriana P. Martinelli Rodriguez
The characterization of cellular changes that occur during somatic embryogenesis is essential for understanding the factors involved in the transition of somatic cells into embryogenically competent cells and determination of cells and/or tissues involved. The present study describes the anatomical and ultrastructural events that lead to the formation of somatic embryos in the model system of the wild passion fruit (Passiflora cincinnata). Mature zygotic embryos were inoculated in Murashige and Skoog induction media supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Zygotic embryo explants at different development stages were collected and processed by conventional methods for studies using light, scanning, and transmission electron microscopy (TEM). Histochemical tests were used to examine the mobilization of reserves. The differentiation of the somatic embryos began in the abaxial side of the cotyledon region. Protuberances were formed from the meristematic proliferation of the epidermal and mesophyll cells. These cells had large nuclei, dense cytoplasm with a predominance of mitochondria, and a few reserve compounds. The protuberances extended throughout the abaxial surface of the cotyledons. The ongoing differentiation of peripheral cells of these structures led to the formation of proembryogenic zones, which, in turn, dedifferentiated into somatic embryos of multicellular origin. In the initial stages of embryogenesis, the epidermal and mesophyll cells showed starch grains and less lipids and protein reserves than the starting explant. These results provide detailed information on anatomical and ultrastructural changes involved in the acquisition of embryogenic competence and embryo differentiation that has been lacking so far in Passiflora. PMID:21927886
Rocha, Diego Ismael; Vieira, Lorena Melo; Tanaka, Francisco André Ossamu; da Silva, Luzimar Campos; Otoni, Wagner Campos
A somatic embryogenesis protocol for plant regeneration of Schisandra chinensis (Turcz.) Baillon was established. Increased callus induction was obtained from mature zygotic embryos on Murashige and Skoog (MS) medium supplemented with 1.0 mg L(-1) N-phenyl-1,2,3-thidiazol-5-yl-urea (TDZ) or 2.0 mg L(-1) 2,4-dichlorophenoxyacetic acid (2,4-D). Addition of Zeatin (Zt) promoted the formation of embryogenic calli. To induce somatic embryogenesis, 2,4-D, TDZ and Zt were incorporated in the medium alone or in combination. Development of the maximum number of somatic embryos (81 globular, 37 heart, 52 torpedo and 37 cotyledon-stage) and germination of the highest number of embryos (50%) was observed on 1/2 MS medium supplemented with 1.0 mg L(-1) TDZ and 0.2 mg L(-1) Zt. Further development of somatic embryos into plantlets was completed in 1/2 MS medium free of plant growth regulators. PMID:24171274
Sun, Dan; Lang, Wen-Xiang; Li, Hong-Bo; Guo, Xiu-Wu; Lian, Mei-Lan; Piao, Zhong-Yun
Significant differences in somatic embryogenesis from melon seeds were observed among 18 cultivars; especially, cultivars ‘Earl's Favorite’ and ‘Barnett’ which produced a large number of somatic embryos. F1 seeds were obtained by reciprocal crosses between cultivars. Some lines produced a large number of somatic embryos whereas others showed no or poor embryogenic response. Most of the F1 seeds formed somatic
Toshiro Oridate; Haruhisa Atsumi; Satishi Ito; Hajime Araki
Following the discovery of somatic embryogenesis in cell cultures of carrot nearly 50 years ago, carrot has served as the primary experimental system to study the molecular biology of this embryogenic episode. Al- though several genes activated or differentiall y expres- sed during somatic embryogenesis in carrot have been identified, the list does not include those critical genes whose regulatory
We established an efficient plant regeneration system for Catharanthus roseus L. (G.) Don through somatic embryogenesis. Embryogenic callus was induced from hypocotyl of seed germinated in vitro. Somatic\\u000a embryogenesis in Catharanthus has been categorized into three distinct stages: (1) initiation and proliferation of embryo; (2) maturation, and; (3) germination\\u000a or plantlet conversion. Beside plant growth regulators, various stages of embryogenesis
Junaid Aslam; A. Mujib; Samar Fatima; M. P. Sharma
A simple method to induce somatic embryogenesis from seeds of rapid-cycling Brassica napus is described. Seedlings cultured on Murashige and Skoog (MS) basal medium produced somatic embryos directly on hypocotyls\\u000a and cotyledons after 2 to 3 subcultures onto the same medium. A low pH of the medium (3.5–5) was more conducive to somatic\\u000a embryogenesis than a higher pH (6 and
W. L. Koh; C. S. Loh
Five genotypes of chickpea (Cicer arietinum L.) PG1, PG5, PG12, N59 and C235 were evaluated for induction of somatic embryogenesis. Somatic embryogenesis was induced from immature cotyledons of genotypes PG12 and C235 and immature embryo axes of genotypes PG5, PG12 and C235. Genotypes N59 and PG1 showed no response. The maximum frequency of globular embryo formation occurred in cotyledonary segments on MS medium with 3.0 mg/l 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Further embryo development was achieved only in somatic embryos derived from cotyledonary segments of genotype PG12. Globular-stage embryos derived from immature embryo axes of PG5, C235, PG12, and cotyledonary segments of C235 dedifferentiated and formed callus. The cotyledonary stage embryos of genotype PG12 germinated on half-strength MS medium supplemented with 1 mg/l zeatin. The regenerated plants were transferred to soil and grown to maturity. PMID:24201882
Sagare, A P; Suhasini, K; Krishnamurthy, K V
Somatic embryogenesis of Cichorium intybus L. var. 'Carolus' is induced using cubical pieces of mature tap roots with an intervening callus phase. A Murashige and Skoog's (MS) semi solid basal medium supplemented with 2,4-dichlorophenoxyacetic acid (0.02 or 0.2 mg/l) and benzylaminopurine (0.25 mg/l) and a liquid MS medium devoid of growth regulators are used respectively for induction of callus and somatic embryoids and for further development and germination. Regeneration from the nodular proembryonal stage to the full grown embryoids occurs following different morphological pathways depending on the physical and chemical environment of the culture. Further development of these embryos into plantlets and the possibilities of application of this technique in plantbreeding have been discussed. PMID:24253697
Heirwegh, K M; Banerjee, N; van Nerum, K; de Langhe, E
Somatic embryogenesis (SE) offers vast potential for the clonal propagation of high-value roses. However, some recalcitrant\\u000a cultivars unresponsive to commonly employed SE-inducing agents and low induction rates currently hinder the commercialization\\u000a of SE technology in rose. Rose SE technology requires improvement before it can be implemented as a production system on a\\u000a commercial scale. In the present work, we assessed
Tammy Estabrooks; Robin Browne; Zhongmin Dong
Summary Somatic embryos of soybean [Glycine max (L.) Merr.] are induced on immature cotyledons explanted onto a medium containing moderately high levels of auxin. Germinability\\u000a of embryos is related to morphologic normality, and both are reduced by excessive exposure to auxin during the induction process.\\u000a Shoot meristem development was improved by reducing exposure of cotyledonary explants from 30 to 10 to
W. A. Parrott; G. Dryden; S. Vogt; D. F. Hildebrand; G. B. Collins; E. G. Williams
Somatic embryogenesis (SE) has the potential to be the lowest-cost method to rapidly produce large numbers of high-value somatic seedlings with desired characteristics for plantation forestry. At least 24 of the 115-120 known Pinus species can undergo SE. Initiation for most species works best with immature megagametophytes as starting material, although a few pines can initiate SE cultures from isolated mature seed embryos. Successful initiation depends heavily on explant type, embryo developmental stage, and medium salt base. Most first reports of initiation used 2,4-D and BAP or a combination of cytokinins. More recent reports have optimized initiation for many Pinus spp., but still use mostly the combinations of auxin and cytokinins. Initiation can be stimulated with medium supplements including abscisic acid (ABA), brassinosteroids, ethylene inhibitors, gibberellin inhibitors, organic acids, putrescine, specific sugar types (maltose, galactose, D-chiro-inositol, and D-xylose), triacontanol, vitamins (B12, biotin, vitamin E, and folic acid), or manipulation of environmental factors including pH, water potential, cone cold storage, gelling agent concentration, and liquid medium. Embryo development and maturation usually occur best on medium containing ABA along with water potential reduction (with sugars and polyethylene glycol) or water availability reduction (with raised gelling agent increasing gel-strength). Activated carbon and maltose may also improve embryo maturation. The main issues holding SE technology back are related to the high cost of producing a somatic seedling, incurred from low initiation percentages for recalcitrant species, culture loss, and decline after initiation and poor embryo maturation resulting in no or poor germination. Although vast progress has been made in pine SE technology over the past 24 years, fundamental studies on seed and embryo physiology, biochemistry, and gene expression are still needed to help improve the technology to a point where large-scale commercialization is economically viable for a broad range of pine species. PMID:21207275
Pullman, Gerald S; Bucalo, Kylie
Arabidopsis LEAFY COTYLEDON (LEC) genes, AtLEC1 and AtLEC2, are important embryonic regulators that play key roles in morphogenesis and maturation phases during embryo development. Ectopic expression of AtLEC1 and AtLEC2 in tobacco caused abnormality in transgenic seedling. When transgenic seeds germinated on medium containing 30 µM DEX, LEC1 transgenic seedlings were ivory and fleshy, with unexpanded cotyledons, stubby hypocotyls, short roots and no obvious callus formation at the shoot meristem position. While LEC2 transgenic seedlings formed embryonic callus on the shoot apical meristem and somatic embryo-like structures emerged from the surface of the callus. When callus were transferred to hormone free MS0 medium more shoots were regenerated from each callus. However, shoot formation was not observed in LEC1 overexpressors. To investigate the mechanisms of LEC2 in somatic embryogenesis, we studied global gene expression by digital gene expression profiling analysis. The results indicated that ectopic expression of LEC2 genes induced accumulation of embryo-specific proteins such as seed storage proteins, late embryogenesis abundant (LEA) proteins, fatty acid biosynthetic enzymes, products of steroid biosynthesis related genes and key regulatory genes of the embryo development. Genes of plant-specific transcription factors such as NAC domain protein, AP2 and GRAS family, resistance-related as well as salicylic acid signaling related genes were up-regulated in LEC2 transgenic seedlings. Ectopi c expression of LEC2 induced large number of somatic embryo formation and shoot regeneration but 20 d DEX induction of LEC1 is not sufficient to induce somatic embryogenesis and shoot formation. Our data provide new information to understand the mechanisms on LEC2 gene's induction of somatic embryogenesis. PMID:23951228
Guo, Fengdan; Liu, Chuanliang; Xia, Han; Bi, Yuping; Zhao, Chuanzhi; Zhao, Shuzhen; Hou, Lei; Li, Fuguang; Wang, Xingjun
Arabidopsis LEAFY COTYLEDON (LEC) genes, AtLEC1 and AtLEC2, are important embryonic regulators that play key roles in morphogenesis and maturation phases during embryo development. Ectopic expression of AtLEC1 and AtLEC2 in tobacco caused abnormality in transgenic seedling. When transgenic seeds germinated on medium containing 30 µM DEX, LEC1 transgenic seedlings were ivory and fleshy, with unexpanded cotyledons, stubby hypocotyls, short roots and no obvious callus formation at the shoot meristem position. While LEC2 transgenic seedlings formed embryonic callus on the shoot apical meristem and somatic embryo-like structures emerged from the surface of the callus. When callus were transferred to hormone free MS0 medium more shoots were regenerated from each callus. However, shoot formation was not observed in LEC1 overexpressors. To investigate the mechanisms of LEC2 in somatic embryogenesis, we studied global gene expression by digital gene expression profiling analysis. The results indicated that ectopic expression of LEC2 genes induced accumulation of embryo-specific proteins such as seed storage proteins, late embryogenesis abundant (LEA) proteins, fatty acid biosynthetic enzymes, products of steroid biosynthesis related genes and key regulatory genes of the embryo development. Genes of plant-specific transcription factors such as NAC domain protein, AP2 and GRAS family, resistance-related as well as salicylic acid signaling related genes were up-regulated in LEC2 transgenic seedlings. Ectopi c expression of LEC2 induced large number of somatic embryo formation and shoot regeneration but 20 d DEX induction of LEC1 is not sufficient to induce somatic embryogenesis and shoot formation. Our data provide new information to understand the mechanisms on LEC2 gene’s induction of somatic embryogenesis.
Xia, Han; Bi, Yuping; Zhao, Chuanzhi; Zhao, Shuzhen; Hou, Lei; Li, Fuguang; Wang, Xingjun
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
Anca Lucau-Danila; Laurent Laborde; Sylvain Legrand; Ludovic Huot; David Hot; Yves Lemoine; Jean-Louis Hilbert; Simon Hawkins; Marie-Christine Quillet; Theo Hendriks; Anne-Sophie Blervacq
Developmental anomalies in the plumule meristem of peanut (Arachis hypogaea L.) somatic embryos resulted in poor shoot differentiation and reduced plant recovery. Existing meristems with caulogenic\\u000a potential have never been tested for embryogenesis in peanut. The present experiment was designed to test the mature zygotic\\u000a embryo axis derived plumule with three meristems for somatic embryogenesis. Embryogenic masses and embryos developed
Shweta Singh; Sulekha Hazra
Somatic embryogenesis-based clonal propagation is suitable to isolate genetically uniform plants of anise (Pimpinella anisum) to develop clonal lines with consistent levels of metabolites like anethole. Somatic embryogenesis in anise was investigated using root explants from two clonal lines in Murashige and Skoog medium with 1 mg\\/liter 2, 4-D and 3 percent sucrose and supplemented with 100 mg\\/liter of fish
Yukiyo Eguchi; John S. Bela; Kalidas Shetty
Cereal embryos can produce two types of callus. One type, termed “embryogenic,” consists of small meristematic-like cells and gives rise to many plants by somatic embryogenesis if placed on a suitable regeneration medium. The other is termed “nonembryogenic” and consists of long tubular cells which gives rise to few or no plants. High concentrations of tryptophan increased the formation of embryogenic callus in three rice cultivars (Oryza sativa L. Calrose 76, Pokkali, and IR 36) but not in four others (Mahsuri, Bg 400-1, H4, and Giza 159). The best concentration of tryptophan for Pokkali and Calrose 76 was 100 micrograms per milliliter, and for IR 36, 50 micrograms per milliliter. Indoleacetic acid at 100 micrograms per milliliter promoted an effect similar to that of tryptophan on Calrose 76. The difference between japonica (Calrose 76, Giza 159) and indica (Pokkali, IR 36) varieties is not the causal factor for the difference in response to tryptophan. Kinetin does not appear to be a requirement for embryogenic callus formation in Calrose 76. Plant regeneration from Calrose 76 embryogenic callus occurred at low levels in media containing no hormones. 6-benzyladenine, or 2,3,5-triiodobenzoic acid but not indoleacetic acid at 0.1 to 0.5 micrograms per milliliter significantly increased regeneration. Images Fig. 1
Siriwardana, Sunitha; Nabors, Murray W.
Summary A procedure for the regeneration of complete plantlets of Tylophora indica from cultured leaf callus via somatic embryogenesis is described. Callus induction from leaf explants was on Murashige and\\u000a Skoog (MS) medium with different concentrations of 2,4-dichlorophenoxyacetic acid (2.4-D; 0.03–3 mg l?1; 0.0–13.56 ?M) and kinetin (Kn; 0.01 mg l?1; 0.05 ?M). The best response for callus induction was obtained
M. Jayanthi; P. K. Mandal
An efficient protocol has been developed for the in vitro propagation of Mexican Weeping Bamboo through somatic embryogenesis from zygotic embryo explants. Mature seeds and excised embryos were cultured in the light or in the dark on both Murashige and Skoog's and Gamborg's B5 basal media with various supplements. Optimal somatic embryogenesis and plant regeneration were obtained by culture in the dark on Murashige and Skoog's basal medium supplemented with 3 mg/1 2,4-dichlorophenoxyacetic acid, 0.5 mg/1 6-benzylaminopurine and 2.0% sucrose. More than 95% of the germinating somatic embryos developed shoots and roots, and were transferred to soil with 85% success. PMID:24203135
Woods, S H; Phillips, G C; Woods, J E; Collins, G B
Somatic embryogenesis is an example of totipotency and is used as a model system for studying embryogenesis. A reproducible tissue culture system was established for the large-scale induction of Arabidopsis somatic embryos. The method allows maintenance of high embryogenic competence over a one-year period. Using this tissue culture system, the expression of embryo-specific genes (ABI3, LEC1, FUS3) was detected in embryogenic cells and somatic embryos. Exogenous application of abscisic acid enhanced the expression of some late-embryogenesis-abundant (LEA) protein genes in somatic embryos. The experiments show that the method can be used to obtain sufficient amounts of embryogenic material for basic molecular analyses. PMID:12096096
Ikeda-Iwai, Miho; Satoh, Shinobu; Kamada, Hiroshi
Summary Somatic embryogenesis and plant regeneration of banana and plantain cultivars (Musa spp.) were obtained by culturing young male flowers. Multiplication and maintenance of embryogenic cultures were achieved\\u000a by culturing somatic embryos in a temporary immersion system (SIT). A multiplication rate of 40 allowed us to obtain more\\u000a than 6000 somatic embryos after 6 mo. of subculture. Plant recovery frequencies were
Jean-Vincent Escalant; Claude Teisson; François Cote
In plants, complete embryos can develop not only from the zygote, but also from somatic cells in tissue culture. How somatic cells undergo the change in fate to become embryogenic is largely unknown. Proteins, secreted into the culture medium such as endochitinases and arabinogalactan proteins (AGPs) are required for somatic embryogenesis. Here we show that carrot (Daucus carota) AGPs can contain glucosamine and N-acetyl-d-glucosaminyl and are sensitive to endochitinase cleavage. To determine the relevance of this observation for embryogenesis, an assay was developed based on the enzymatic removal of the cell wall from cultured cells. The resulting protoplasts had a reduced capacity for somatic embryogenesis, which could be partially restored by adding endochitinases to the protoplasts. AGPs from culture medium or from immature seeds could fully restore or even increase embryogenesis. AGPs pretreated with chitinases were more active than untreated molecules and required an intact carbohydrate constituent for activity. AGPs were only capable of promoting embryogenesis from protoplasts in a short period preceding cell wall reformation. Apart from the increase in embryogenesis, AGPs can reinitiate cell division in a subpopulation of otherwise non-dividing protoplasts. These results show that chitinase-modified AGPs are extracellular matrix molecules able to control or maintain plant cell fate.
van Hengel, Arjon J.; Tadesse, Zewdie; Immerzeel, Peter; Schols, Henk; van Kammen, Ab; de Vries, Sacco C.
Embryogenic Daucus carota L. cells grown in 9 micromolar 2.4-dichlorophenoxyacetic acid are resistant to greater than 5 micromolar 5-bromodeoxyuridine (BrdU). In contrast, 5 micromolar BrdU strongly inhibits somatic embryogenesis within 24 hours after transfer of cells to an auxin-free medium. DNA synthesis rates in control and BrdU-treated cultures are rapid and similar; however, the DNA content does not reach levels as great in the presence of BrdU as in control cultures. BrdU substitutes for thymidine in the DNA in 28% of the available sites 48 hours after auxin removal. Following DNA repair, somatic embryogenesis resumes. BrdU DNA incorporation leads to somatic embryogenesis inhibition and provides an alternative to auxin treatment for the interruption of carrot cell culture differentiation. Images Figure 2 Figure 3 Figure 7
Thomas, John C.; Nessler, Craig; Katterman, Frank
Culture of Citrus sinensis embryogenic callus on the embryo-inducing medium (EIM) containing glycerol gave rise to a large number of embryos, whereas very few embryos were observed on the callus growth medium (CGM). In the current paper, attempts were made to investigate whether polyamine biosynthesis was involved in glycerol-mediated somatic embryogenesis. Quantification of free polyamines by high-performance liquid chromatography showed that the cultures on EIM had less putrescine than those on CGM. However, increase in spermidine and spermine was detected in cultures on EIM during the first 20d of culture, coincident with abundant somatic embryogenesis. The globular embryos contained more polyamines than embryos at other stages. Semi-quantitative reverse transcriptase-polymerase chain reaction assay showed that expression levels of all of the five key genes involved in polyamine biosynthesis, with the exception of S-adenosylmethionine decarboxylase, were induced in cultures on EIM, and that their transcriptional levels were increased with maturation of the embryos. Addition of alpha-difluoromethylornithine, a polyamine biosynthesis inhibitor, to EIM resulted in remarkable inhibition of somatic embryogenesis, concurrent with notable reduction of endogenous putrescine and spermidine, particularly at higher concentrations. Exogenous application of 1mM putrescine to EIM together with 5mM alpha-difluoromethylornithine led to dramatic enhancement of endogenous polyamines, which successfully restored somatic embryogenesis. All of these, collectively, demonstrated that free polyamines, at least spermidine and spermine herein, were involved in glycerol-mediated promotion of somatic embryogenesis, which will open a new avenue for establishing a sophisticated system for somatic embryogenesis based on the modulation of endogenous polyamines. PMID:18448195
Wu, Xiao-Ba; Wang, Jing; Liu, Ji-Hong; Deng, Xiu-Xin
In monocots, the zygotic embryo is protected and nourished by an endosperm. In the present study starch deposition and amylase accumulation was noticed during somatic embryogenesis in stem callus of a bamboo, Dendrocalamus hamiltonii. SEM studies revealed that starch grains were clearly visible in the scutellum during the maturation stage of the somatic embryo. As the somatic embryo developed further, the scutellum got reduced with corresponding increase in amylase. The amylase activity was tested periodically at different developmental stages of embryos. The role of scutellum in somatic embryos for starch deposition and amylase accumulation is discussed. PMID:15022840
Godbole, Savita; Sood, Anil; Sharma, Madhu; Nagar, Pramod Kumar; Ahuja, Paramvir Singh
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  showed that the use of the ?-D-glucosyl Yariv reagent (?-GlcY) that specifically binds arabinogalactan-proteins (AGPs) blocked somatic embryo production in chicory root explants. This observation indicates that ?-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 . 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 ?-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. ?-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 ?-GlcY-treatment. Eight genes were both differentially expressed between K59 and C15 genotypes during SE induction and transcriptionally affected by ?-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 ?-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.
Somatic embryogenesis receptor-like kinase (SERK) belonging to the receptor-like kinases (RLKs) has been shown to be implicated in somatic embryogenesis (SE). In this study, a somatic embryogenesis receptor-like\\u000a gene CitSERK1-like was cloned and characterized from Citrus sinensis cv. ‘Valencia’, a genotype with high somatic embryogenesis capacity for over 26 years. Fifteen consecutive amino acids in\\u000a putative leucine zipper domain of CitSERK1-like
Xiao-Xia Ge; Gai-En Fan; Li-Jun Chai; Wen-Wu Guo
Four systems of cyclic somatic embryogenesis of Siberian ginseng (Eleutherococcus senticosus Maxim) were used to study the mechanism of embryonic cell cluster generation. The first, direct somatic embryo induction (DSEI), generates secondary embryos directly from the primary somatic embryos; the second, direct embryogenic cell cluster induction (DEC)), induces embryogenic cell clusters directly from somatic embryos in agar medium. Subsequently, we found that when DEC-derived somatic embryos are transferred to suspension culture or a bioreactor culture, only somatic embryos are induced, and embryogenic cell clusters cannot form. Therefore, these new lines were named DEC cultured by liquid medium (ECS) and DEC cultured by bioreactor (ECB), respectively. Transmission electron microscopy showed that DEC epidermal cells contained a variety of inclusions, distinct from other lines. A cDNA library of DEC was constructed, and 1,948 gene clusters were obtained and used as probes. RNA was prepared from somatic embryos from each of the four lines and hybridized to a microarray. In DEC, 7 genes were specifically upregulated compared with the other three lines, and 4 genes were downregulated. EsXTH1 and EsPLT1, which were among the genes upregulated in DEC, were cloned using the rapid amplification of cDNA ends (RACE). Real-time quantitative PCR showed EsXTH1 was more highly expressed in DEC than in other lines throughout the culture cycle, and EsPLT1 expression in DEC increased as culture duration increased, but remained at a low expression level in other lines. These results suggest that EsXTH1 and EsPLT1 may be the essential genes that play important roles during the induction of embryogenic cell clusters. PMID:24743225
Zhou, Chenguang; Liu, Likun; Li, Chenghao
An efficient somatic embryogenesis system has been established in six date palm (Phoenix dactylifera L.) cultivars (Barhee, Zardai, Khalasah, Muzati, Shishi and Zart). Somatic embryogenesis (SE) was growth regulators and cultivars dependent. Friable embryogenic callus was induced from excised shoot tips on MS medium supplemented with various auxins particularly 2,4-dichlorophenoxyacetic acid (2,4-D, 1.5 mg 1(-l)). Suspension culture increased embryogenesis potentiality. Only a-naphthaleneacetic acid (NAA, 0.5 mg 1(-1)) produced somatic embryos in culture. Somatic embryos germinated and converted into plantlets in N(6)-benzyladenine (BAP, 0.75 mg 1(-l)) added medium following a treatment with thidiazuron (TDZ, 1.0 mg 1(-l)) for maturation. Scanning electron microscopy showed early stages of somatic embryo particularly, globular types, and was in masses. Different developing stages of embryogenesis (heart, torpedo and cotyledonary) were observed under histological preparation of embryogenic callus. Biochemical screening at various stages of somatic embryogenesis (embryogenic callus, somatic embryos, matured, germinated embryos and converted plantlets) of date palm cultivars has been conducted and discussed in detail. The result discussed in this paper indicates that somatic embryos were produced in numbers and converted plantlets can be used as a good source of alternative propagation. Genetic modification to the embryo precursor cell may improve the fruit quality and yield further. PMID:23961149
Aslam, Junaid; Khan, Saeed Ahmad; Cheruth, Abdul Jaleel; Mujib, Abdul; Sharma, Maheshwar Pershad; Srivastava, Prem Shanker
We developed a new protocol for highly efficient somatic embryogenesis and plantlet conversion of Schisandra chinensis. Friable embryogenic callus was induced from cotyledonary leaves and hypocotyls of germinated zygotic embryos on Murashige\\u000a and Skoog (MS) agar medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). Preculture of zygotic embryos on 2,4-D-containing\\u000a medium increased embryogenic callus induction efficiency. The highest embryogenic callus induction frequency of
Ai Hua Chen; Jing Li Yang; Yu Da Niu; Chuan Ping Yang; Gui Feng Liu; Chang Yeon Yu; Cheng Hao Li
Detailed expression analysis of the Norway spruce (Picea abies (L.) Karst) Viviparous 1 (Pavp1) and p34cdc2 (cdc2Pa) genes was carried out during somatic embryogenesis. Pavp1, a gene associated with embryo development, was expressed in prolifer- ating embryogenic suspension cultures in the absence of exogenous ABA. When somatic embryo formation was promoting by blocking proliferation, Pavp1 expression was reduced. During maturation,
Steven Footitt; Mathieu Ingouff; David Clapham; Sara von Arnold
Messenger RNAs from cultures of embryogenic and non-embryogenic alfalfa (Medicago sativa L.) genotypes were used to differentially screen a cDNA library prepared from embryogenic cell masses of somatic embryo cultures to identify early-stage embryo transcripts. The three alfalfa somatic embryogenesis-specific transcripts cDNAs (ASET1, ASET2 and ASET3) identified by this screen were enriched in RNA samples from embryogenic tissues of the
R. W. Giroux; K. P. Pauls
An efficient procedure has been developed for inducing somatic embryogenesis from mature leaves of Tylophora indica (Burm.f.) Merrill, an important medicinal plant. Leaf sections were initially cultured on Murashige and Skoog's (MS) medium supplemented with thidiazuron (TDZ) in addition with 2, 4-dichlorophenoxy acetic acid (2,4-D), particularly 0.5 µm TDZ along with 1.5 µm 2,4-D was very effective in inducing somatic
T. Chandrasekhar; T. Mohammad Hussain; G. Rama Gopal; J. V. Srinivasa Rao
Mature embryos of Acanthopanax senticosus explanted on Murashige and Skoog (MS) medium with 0.5 mg\\/1 2,4-D developed somatic embryos directly from swollen cotyledon and embryo axes within one to two months. When the somatic embryos were transferred to medium supplemented with 2,4-D (0.5 mg\\/1) or IAA (1–3 mg\\/1) or Zeatin (0.5 mg\\/1) and NAA (0.2 mg\\/1), additional somatic embryos developed.
Y. Gui; Z. Guo; S. Ke; R. M. Skirvin
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.
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
We report, an efficient protocol for plantlet regeneration from the cell suspension cultures of cowpea through somatic embryogenesis. Primary leaf-derived, embryogenic calli initiated in MMS [MS salts (Murashige and Skoog 1962) with B5 (Gamborg et al. 1968) vitamins] medium containing 2,4-Dichlorophenoxyacetic acid (2,4-D), casein hydrolysate (CH), and
K. Ramakrishnan; R. Gnanam; P. Sivakumar; A. Manickam
A method for plant regeneration via somatic embryogenesis was established in aloe (Aloe barbadensis Mill.). For explant disinfection, treatments involved 2, 3, 4, 5, 10 and 15 min sonication, in combination with 4% v\\/v NaOCl. Explant source and growth regulators were investigated. The highest survival rate (85%) and the lowest contamination (15%) were obtained with 5 min sonication. Friable embryogenic
Giovanni Garro-Monge; Andrés M. Gatica-Arias; Marta Valdez-Melara
Most of the plant regeneration processes in citrus, through tissue culture, involve somatic embryogenesis. The optimization of these processes is important for the development of in vitro plant improvement. Nodal segments and leaf discs of sweet orange (Citrus sinensis L.) cv. Musambi and Lime (Citrus aurantifolia ) cv. Kaghzi Nimbu were used to obtain aseptically raised plantlets of Lime (Citrus
RASHAD MUKHTAR; M. MUMTAZ KHAN; RAMZAN RAFIQ; ADNAN SHAHID; FAROOQ AHMAD KHAN
Various tissues of seeds and seedlings of melon were cultured in vitro to study the effects of auxin concentration on organogenesis and embryogenesis. Adventitious shoots and somatic embryos were formed from explants of cotyledons of mature seeds, hypocotyls of seedlings, and leaves and petioles of young plantlets. Expanded cotyledons of seedlings formed only adventitious shoots. All tissues responded similarly to
Yutaka Tabei; Tsuguo Kanno; Takeshi Nishio
Mature embryos of Acanthopanax senticosus explanted on Murashige and Skoog (MS) medium with 0.5 mg/1 2,4-D developed somatic embryos directly from swollen cotyledon and embryo axes within one to two months. When the somatic embryos were transferred to medium supplemented with 2,4-D (0.5 mg/1) or IAA (1-3 mg/1) or Zeatin (0.5 mg/1) and NAA (0.2 mg/1), additional somatic embryos developed. Most (93%) embryos germinated on the above medium without 2,4-D. Sixty-two percent of the plantlets survived in soil. Histological observations revealed that the somatic embryos originated from cell masses of epidermal and sub-epidermal origin. There was no cytological separation zone between the somatic embryos and cultured expiants. Consequently, embryos were difficult to separate from their expiant tissue. PMID:24213792
Gui, Y; Guo, Z; Ke, S; Skirvin, R M
Plant regeneration through somatic embryogenesis from young leaf explants (5–10 mm long) adjacent to the apex of 5–6 year\\u000a old offshoots of Tunisian date palm (Phœnix dactylifera L.), cultivar Boufeggous was successfully achieved. Factors affecting embryogenic callus initiation, including plant growth\\u000a regulators and explant size, were investigated. The highest induction frequencies of embryogenic calli occurred after 6–7 months\\u000a on MS medium supplemented with
A. Othmani; C. Bayoudh; N. Drira; M. Marrakchi; M. Trifi
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)-?-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.
Xu, Chunxiang; Zhao, Lu; Pan, Xiao; Samaj, Jozef
In southern and south-western Ethiopia, Ensete ventricosum is grown as an important starchy, staple food crop, supporting the diet of a quarter of the Ethiopian population. Due to difficulty in germinating seeds and the long vegetative period, breeding enset is extremely difficult. Adventitious buds and somatic embryos have been induced from callus derived from corm tissues and cultured on Murashige and Skoog's (MS) basal medium supplemented with benzylaminopurine (BAP) or 6 ?-?-dimethylallylamino purine 2iP. Elongation of somatic embryos was achieved on the same medium and rooting was induced on half-strength MS basal medium supplemented with IBA. No phenotypic variation was observed among more than 200 potted regenerants. The possible implications for mutation breeding in this crop are discussed. PMID:24178427
Afza, R; van Duren, M; Morpnrgo, R
An efficient plant regeneration procedure has been established from hypocotyl explants of the common ice plant, Mesembryanthemum crystallinum L, a halophytic leaf succulent that exhibits a stress-induced switch from C3 photosynthesis to crassulacean acid metabolism\\u000a (CAM). Somatic embryos were initiated and developed up to globular and heart stages in Murashige and Skoog (MS) media supplemented\\u000a with 3% sucrose, 0.6% bacto-agar,
J. C. Cushman; T. Wulan; N. Kuscuoglu; M. D. Spatz
Breeding and selection was used to generate a population of white clover (Trifolium repens L.) from cultivar Osceola with a high embryogenic capacity. Somatic embryos were obtained from immature cotyledons of white clover placed onto EC6 basal medium containing 40 mg L-1 of 2,4-D and 6% sucrose. The effects of 2,4-D at 20 and 40 mg L-1 and of the
A. Keyton Weissinger; Wayne A. Parrott
High frequency somatic embryogenesis in papaya (Carica papaya L.) tissue cultures was achieved by culturing hypocotyl sections from ten-day-old seedlings on half-strength Murashige and Skoog salts (MS) medium containing modified MS vitamins, 2.3 to 112.5 µM 2,4-dichlorophenoxyacetic acid (2,4-d), 400 mg l-1 glutamine, and 6% sucrose. Four hermaphroditic Hawaiian cultivars produced embryogenic calluses after ten to 14 weeks of culture
Maureen M. M. Fitch
The Arabidopsis thaliana Somatic Embryogenesis Receptor Kinases (SERKs) consist of five members, SERK1 to SERK5, of the leucine-rich repeat receptor-like kinase subfamily II (LRR-RLK II). SERK3 was named BRI1-Associated Receptor Kinase 1 (BAK1) due to its direct interaction with the brassinosteroid (BR) receptor BRI1 in vivo, while SERK4 has also been designated as BAK1-Like 1 (BKK1) for its functionally redundant
Xiaoping Gou; Hongju Yin; Kai He; Junbo Du; Jing Yi; Shengbao Xu; Honghui Lin; Steven D. Clouse; Jia Li
Summary This study was conducted to examine the effect of biotin and thiamine concentrations on callus growth and somatic embryogenesis\\u000a of date palm (Phoenix dactylifera L.). Embryogenic callus derived from offshoot tip explants was cultured on hormone-free MS medium containing biotin at 0,\\u000a 0.1, 1, or 2 mg l?1 combined with thiamine at 0.1, 0.5, 2, or 5 mg l?1. Embryogenic
Jameel M. Al-Khayri
Plants were obtained via somatic embryogenesis in callus derived from in vitro raised leaf and petiole explants of Aconitum heterophyllum Wall. Callus was induced on a Murashige-Skoog medium supplemented with either 2,4-dichlorophenoxy acetic acid (2,4-d 1 mg l-1) and kinetin (KN 0.5 mg l-1) with coconut water (CW 10% v\\/v) or naphthalene acetic acid (NAA 5 mg l-1) and benzylaminopurine
Archana Giri; Paramvir Singh Ahuja; P. V. Ajay Kumar
Root segments from spinach (Spinacia oleracea L. cv. Jiromaru) seedlings form embryogenic callus (EC) that responded to exogenous GA(3) by accumulating a 31-kDa glycoprotein [BP31 or S. oleracea ribosome-inactivating protein (EC 126.96.36.199) (SoRIP1)] in association with the expression of embryogenic potential. Microsequencing of this protein revealed significant similarity with type 1 RIPs. We identified cDNAs for SoRIP1 and S. oleracea RIP2 (SoRIP2), a novel RIP having a consensus shiga/ricin toxic domain and performed a comparative analysis of the expression of SoRIPs during somatic embryogenesis. Western blotting and quantitative polymerase chain reaction analyses revealed that the expression of SoRIP1 in calli increased remarkably in association with the acquisition of embryogenic potential, although the expression in somatic embryos decreased moderately with their development. However, the expression of SoRIP2 in calli remained low and constant but increased markedly with the development of somatic embryos. Treatment of callus with GA(3) and/or ABA for 24 h, or with ABA for a longer period, failed to stimulate the expression of either gene. Immunohistochemistry showed that SoRIP1 preferentially accumulated in the proembryos and peripheral meristem of somatic embryos early in development. Appreciable expression of SoRIP2 was not detected in the callus, but intense expression was found in the epidermis of somatic embryos. These results suggest that the expression of spinach RIP genes is differentially regulated in a development-dependent fashion during somatic embryogenesis in spinach. PMID:18494862
Kawade, Kensuke; Ishizaki, Takuma; Masuda, Kiyoshi
Somatic embryogenesis has been described in peach palm as a reliable method for its in vitro multiplication and conservation. In this study, we evaluated the possible role of arabinogalactan proteins (AGPs) during this morphogenetic pathway. The presence of Yariv reagent, a synthesized chemical antibody that specifically binds AGP molecules, affected somatic embryos and callus development rate, but no effect was observed on fresh weight increment. This substance also had profound effects on embryo morphology: somatic embryos presented loose cells in the protoderm and no signs of polarization could be observed. To better evaluate the role of AGPs, analyses of specific monoclonal antibodies (MAbs) against different AGP epitopes revealed a specific pattern of distribution for each epitope. MAb JIM13 had differential expression and showed intense signal on the embryogenic sector and some immediately adjacent layers. MAb JIM7 against pectin recognized cell walls and a specific layer over the developing somatic embryo, as well as over the shoot meristem region of mature somatic embryos. This corresponds to an extracellular matrix surface network (ECMSN) associated with the development of somatic embryos and closely related to the expression of MAb JIM13. Scanning electron microscopy confirmed the presence of an ECMSN covering a specific group of cells and ultra-structural analyses revealed that the ECMSN had lipophilic substances. PMID:22574975
Steinmacher, Douglas A; Saare-Surminski, Katja; Lieberei, Reinhard
Cultured asparagus (Asparagus officinalis L. cv Y6) cells induced to regenerate into whole plants through somatic embryogenesis secreted a 38-kD protein into cell walls. The full-length cDNA sequence of this protein (Asparagus officinalis peroxidase 1 [AoPOX1]) determined by reverse transcriptase-polymerase chain reaction showed similarity with plant peroxidases. AoPOX1 transcripts were particularly abundant during early somatic embryogenesis. To evaluate the in vivo function of AoPOX1 protein, purified recombinant AoPOX1 protein was reacted with a series of phenolic substrates. The AoPOX1 protein was effective in the metabolism of feruloyl (o-methoxyphenol)-substituted substrates, including coniferyl alcohol. The reaction product of coniferyl alcohol was fractionated and subjected to gas chromatography-mass spectrometry analysis and 1H-nuclear magnetic resonance analysis, indicating that the oxidation product of coniferyl alcohol in the presence of AoPOX1 was dehydrodiconiferyl alcohol. The concentration of dehydrodiconiferyl alcohol in the cultured medium of the somatic embryos was in the range of 10?8 m. Functions of the AoPOX1 protein in the cell differentiation are discussed.
Takeda, Hiroyuki; Kotake, Toshihisa; Nakagawa, Naoki; Sakurai, Naoki; Nevins, Donald J.
We have located a novel carbohydrate epitope in the cell walls of certain single cells in embryogenic, but not in non-embryogenic, suspension cultures of carrot. Expression of this epitope, recognized by the mAb JIM8, is regulated during initiation, proliferation, and prolonged growth of suspension cultures such that changes in the abundance of JIM8-reactive cells always precede equivalent changes in embryogenic potential. Therefore, a direct correlation exists between the presence of the JIM8-reactive cell wall epitope and somatic embryo formation. The JIM8-reactive cell wall epitope is expressed in the cell walls of three types of single cells and one type of cell cluster. One of the single cell types seems able to follow one of two phytohormone- controlled developmental pathways, either a cell elongation pathway that eventually leads to cell death, or a cell division pathway that gives rise to proembryogenic masses. We demonstrate that all JIM8- reactive cell types in embryogenic carrot suspension cultures are developmentally related, and that the switch by one of them to somatic embryogenesis is accompanied by the immediate dissipation of the JIM8- reactive cell wall epitope. The cell wall carbohydrate epitope recognized by JIM8 therefore represents a cell wall marker for a very early transitional cell state in the developmental pathway to carrot somatic embryogenesis.
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. PMID:21496030
Pan, Xiao; Yang, Xiao; Lin, Guimei; Zou, Ru; Chen, Houbin; Samaj, Jozef; Xu, Chunxiang
Somatic embryogenesis was achieved from cell suspension cultures of niger (Guizotia abyssinica Cass.). Initially, friable embryogenic calluses were induced from cotyledonary leaves of niger on Murashige and Skoog (MS)\\u000a agar medium containing 5 ?M 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.5 ?M kinetin (KIN). Cell suspension cultures were\\u000a established by using embryogenic calluses in MS liquid medium containing 5 ?M 2,4-D and 0.5 ?M KIN. Initiation
Poornananda Madhava Naik; Hosakatte Niranjana Murthy
Plant regeneration and somatic embryogenesis through interspecific hybridization among different Carica species were studied for the development of a papaya ringspot virus-resistant variety. The maximum fruit sets were recorded from the cross of the native variety C. papaya cv. Shahi with the wild species C. cauliflora. The highest hybrid embryos were recorded at 90 days after pollination and the embryos were aborted at 150 days after pollination. The immature hybrid embryos were used for plant regeneration and somatic embryogenesis. The 90-day-old hybrid embryos from the cross of C. papaya cv. Shahi × C. cauliflora showed the highest percentage of germination, as well as plant regeneration on growth regulators free culture medium after 7 days pre-incubation on half-strength MS medium supplemented with 0.2 mg/L BAP, 0.5 mg/L NAA and 60 g/L sucrose. The 90-day-old hybrid embryos from the cross of C. papaya cv. Shahi × C. cauliflora produced maximum callus, as well as somatic embryos when cultured on half-strength MS medium containing 5 mg/L 2,4-D, 100 mg/L glutamine, 100 mg/L casein hydrolysate and 60 g/L sucrose. The somatic embryos were transferred into half-strength MS medium containing 0.5 mg/L BAP and 0.2 mg/L NAA and 60 g/L sucrose for maturation. The highest number of regenerated plants per hybrid embryo (10.33) was recorded from the cross of C. papaya cv. Shahi × C. cauliflora. Isoenzyme and dendrogram cluster analysis using UPGMA of the regenerated F(1) plantlets confirmed the presence of the hybrid plantlets. PMID:23235330
Azad, Md Abul Kalam; Rabbani, Md Golam; Amin, Latifah
In the present paper, a method for the induction and long-term maintenance embryogenic cultures for Vitis Labruscana `Niagara' and `Fredonia' is reported. Embryogenic cultures from these two cultivars were induced in an embryogenesis establishment\\u000a medium (CIM) from ovaries obtained from flowers 10–14 days pre-anthesis. The embryogenic lines obtained in this experiment\\u000a have been stably maintained for more than 2 years,
S. Y. Motoike; R. M. Skirvin; M. A. Norton; A. G. Otterbacher
• Background and Aims The date palm is a dioecious perennial species of the Arecaceae for which in vitro micropropagation is essential to ensure the renewal of palm plantations. This study presents a histocytological analysis of the traditional Mauritanian Amsekhsi cultivar beginning from the initial callogenesis and continuing up to the establishment of the cellular embryogenic cell suspensions. The formation of somatic embryos and their development into rooted plants are also described. • Methods Foliar segments of seedlings cultured in the presence of 2,4-D produced primary calli that were chopped to produce fine friable granular calli that subsequently produced cellular suspensions when transferred to liquid medium. The somatic proembryos that developed after removal of the 2,4-D were plated on agar medium where they developed into rooted plants. Thin sections of tissue fragments taken at each stage of the process were stained using Periodic Acid Schiff and Naphthol Blue-Black. • Key Results The first cellular divisions were localized close to the vascular vessels of the leaf. The primary calli were obtained within 2 months. Fine friable granular calli grew quickly after the primary calli were chopped. Individual embryogenic cells were identified that rapidly started to divide and developed into globular proembryos. In addition, in the microcalli, breaking zones appeared in the thick pectocellulosic walls which delimited the pluricellular proembryos. The anatomy of somatic embryos is similar to that of zygotic embryos despite a deficit in the accumulation of intracellular proteins. When rooted with NAA, the vitroplants developed a strong orthotropic taproot. • Conclusions This study contributes to understanding the whole process of somatic embryogenesis, but two specific questions remain to be answered: what factors are involved in the reactivation of the somatic cells at the beginning of the initial callogenesis, and why do the somatic embryos not accumulate proteins in their tissues during maturation?
SANE, D.; ABERLENC-BERTOSSI, F.; GASSAMA-DIA, Y. K.; SAGNA, M.; TROUSLOT, M. F.; DUVAL, Y.; BORGEL, A.
An efficient method has been developed for regeneration of complete plants via somatic embryogenesis in Corydalis yanhusuo (Fumariaceae), an important medicinal plant, using tuber-derived callus. Primary callus was induced by culturing mature tuber pieces on Murashige and Skoog's (MS) medium supplemented with 2.0 mg l(-1) N(6)-benzyladenine (BA) and 0.5 mg l(-1) alpha-naphthaleneacetic acid (NAA) in darkness. Somatic embryos were induced by subculturing the primary callus on MS medium supplemented with 0.5-4.0 mg l(-1) BA, kinetin, or zeatin, within 2 weeks of culture in light. Embryos with well-developed cotyledonary leaves were transferred in half-strength liquid MS medium supplemented with 1.0 mg l(-1) zeatin riboside for the development of roots. Converted somatic embryos were cultured on half-strength MS medium supplemented with 6% sucrose, and with 0.5-10.0 mg l(-1) abscisic acid (ABA), paclobutrazol, or ancymidol, 0.5-5.0 mg l(-1) GA(3) and 15-100 mg l(-1) polyethylene glycol (PEG) 4000 for further development of plantlets and in vitro tuber formation. The development of somatic embryos over the surface of tuber and/or cotyledonary leaf base region of the converted primary somatic embryo was observed. Before ex vitro establishment of somatic embryo-derived plants, plants with well-developed tubers were cultured on half-strength MS medium with 2% sucrose and 0.1 mg l(-1) GA(3) for 3 weeks. PMID:11164586
Sagare, A P.; Lee, Y L.; Lin, T C.; Chen, C C.; Tsay, H S.
The effect of sugars, gibberellic acid (GA3) and abscisic acid (ABA) on somatic embryogenesis from internodal explant-derived callus of Tylophora indica (Burm. f.) Merrill has been investigated. Embryogenic calli were produced from internodal explants and the best result was achieved by using MS medium supplemented with 4micromol/L 2, 4-Dichlorophenoxyacetic acid (2, 4-D). Up to 69% of such embryogenic calli differentiated into somatic embryos with an average of 25 embryos per explant (per gram of the calli) on Murashige and Skoog (MS) medium containing 6micromol/L kinetin (Kn). The individual effect of sucrose and glucose together with 6micromol/L Kn was evaluated. There was a significant difference among concentrations of sugar and among kinds of sugar tested in somatic embryogenesis. Sucrose at 200mmol/L with 6micromol/L Kn gave rise to a maximum embryogenesis (71%) with an average of 49 embryos per explant. However, glucose together with 6micromol/L Kn or a combination of glucose, sucrose and 6micromol/L Kn reduced the percentage of embryogenesis culture and the number of embryos per explant. The presence of GA3 and ABA at particular concentrations promoted somatic embryogenesis in T. indica. The addition of 10mol/L GA3 into the 200mmol/L sucrose-containing medium gave a 98% embryogenesis response with an average of 51 embryos per explant. Somatic embryogenesis was significantly enhanced by the addition of 2micromol/L ABA to 200mmol/L sucrose-containing medium. On this medium 95% embryogenesis with an average of 44 embryos per explant was observed. The study reported here indicates that 200mmol/L sucrose with 6micromol/L Kn, 200mmol/L sucrose with 10micromol/L GA3 and 200mmol/L sucrose with 2micromol/L ABA significantly improved somatic embryogenesis in T. indica whereas glucose alone or in combination with sucrose had an inhibitory role. The embryos obtained developed normally and were easily converted into plants. PMID:16755928
Thomas, Dennis T
Expression of the Agrobacterium rhizogenes rolC gene in Panax ginseng callus cells results in formation of tumors that are capable to form roots. The selection of non-root forming tumor clusters yielded the embryogenic 2c3 callus line, which formed somatic embryos and shoots independently of external growth factors. Although the 2c3 somatic embryos developed through a typical embryogenesis process, they terminated prematurely and repeatedly formed adventitious shoot meristems and embryo-like structures. A part of the shoots and somatic embryos formed enlarged and fasciated meristems. This is the first indication of the rolC gene embryogenic effect and, to our knowledge, the first indication that a single gene of non-plant origin can induce somatic embryogenesis in plants. PMID:16136334
Gorpenchenko, T Y; Kiselev, K V; Bulgakov, V P; Tchernoded, G K; Bragina, E A; Khodakovskaya, M V; Koren, O G; Batygina, T B; Zhuravlev, Yu N
Many of the regulatory processes occurring during plant embryogenesis are still unknown. Relatively few cells are involved, and they are embedded within maternal tissues, making this developmental phase difficult to study. Somatic embryogenesis is a more accessible system, and many important regulatory genes appear to function similar to zygotic development, making somatic embryogenesis a valuable model for the study of zygotic processes. To better understand the role of the Arabidopsis (Arabidopsis thaliana) MADS factor AGAMOUS-Like15 (AGL15) in the promotion of somatic embryogenesis, direct target genes were identified by chromatin immunoprecipitation-tiling arrays and expression arrays. One potential directly up-regulated target was At5g61590, which encodes a member of the ethylene response factor subfamily B-3 of APETALA2/ETHYLENE RESPONSE FACTOR transcription factors and is related to Medicago truncatula SOMATIC EMBRYO-RELATED FACTOR1 (MtSERF1), which has been shown to be required for somatic embryogenesis in M. truncatula. Here, we report confirmation that At5g61590 is a directly expressed target of AGL15 and that At5g61590 is essential for AGL15’s promotion of somatic embryogenesis. Because At5g61590 is a member of the ETHYLENE RESPONSE FACTOR family, effects of ethylene on somatic embryogenesis were investigated. Precursors to ethylene stimulate somatic embryogenesis, whereas inhibitors of ethylene synthesis or perception reduce somatic embryogenesis. To extend findings to a crop plant, we investigated the effects of ethylene on somatic embryogenesis in soybean (Glycine max). Furthermore, we found that a potential ortholog of AGL15 in soybean (GmAGL15) up-regulates ethylene biosynthesis and response, including direct regulation of soybean orthologs of At5g61590/MtSERF1 named here GmSERF1 and GmSERF2, in concordance with the M. truncatula nomenclature.
Zheng, Qiaolin; Zheng, Yumei; Perry, Sharyn E.
The effect of explant age, plant growth regulators and culture conditions on somatic embryogenesis and rosmarinic acid production\\u000a from leaf explants of Salvia officinalis and S. fruticosa plants collected in Greece was investigated. Embryogenic callus with numerous spherical somatic embryos could be induced\\u000a on explants derived from both species and cultured for 3 weeks on a Murashige and Skoog (MS)
S. Kintzios; A. Nikolaou; M. Skoula
Brassinosteroids (BRs) are key regulators in plant growth and development. The main BR-perceiving receptor in Arabidopsis (Arabidopsis thaliana) is BRASSINOSTEROID INSENSITIVE1 (BRI1). Seedling root growth and hypocotyl elongation can be accurately predicted using a model for BRI1 receptor activity. Genetic evidence shows that non-ligand-binding coreceptors of the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family are essential for BRI1 signal transduction. A relatively simple biochemical model based on the properties of SERK loss-of-function alleles explains complex physiological responses of the BRI1-mediated BR pathway. The model uses BRI1-BR occupancy as the central estimated parameter and includes BRI1-SERK interaction based on mass action kinetics and accurately describes wild-type root growth and hypocotyl elongation. Simulation studies suggest that the SERK coreceptors primarily act to increase the magnitude of the BRI1 signal. The model predicts that only a small number of active BRI1-SERK complexes are required to carry out BR signaling at physiological ligand concentration. Finally, when calibrated with single mutants, the model predicts that roots of the serk1serk3 double mutant are almost completely brassinolide (BL) insensitive, while the double mutant hypocotyls remain sensitive. This points to residual BRI1 signaling or to a different coreceptor requirement in shoots. PMID:24072582
van Esse, Wilma; van Mourik, Simon; Albrecht, Catherine; van Leeuwen, Jelle; de Vries, Sacco
Complex organic additives are known to improve growth and differentiation of in vitro plant cultures. The present investigation was conducted to determine the effect of various concentrations of yeast extract (YE) and casein hydrolysate (CH) on callus growth and somatic embryogenesis in date palm cultivar Nabout Saif. Callus induced from shoot tip explants was grown on callus multiplication medium supplemented
Jameel M. Al-Khayri
The complexity of the somatic embryogenesis (SE) transcriptome suggests that numerous molecules are involved. To understand better the functional genomics of complex molecular systems during this important reprogramming process, we used bioinformatics and a pathway database to construct a draft network based on transcriptionally regulated SE-related genes, from functional genomics assays readout to high-level biological data interpretation. Here, a complex molecular system was unraveled by this network. This draft network is a potential reservoir for hundreds of testable predictions about cellular processes in early SE. This work could provide a useful test for modeling of a systems network and may have merit as a study presenting an advanced technology application due to its biological and economical importance. The approach presented here is scalable and can be extended to include additional data types. In particular, this effective system approach will be applied to various targeted gene networks in the future. PMID:17884330
Zeng, Fanchang; Zhang, Xianlong; Cheng, Lei; Hu, Lisong; Zhu, Longfu; Cao, Jinglin; Guo, Xiaoping
Brassinosteroids, which control plant growth and development, are sensed by the leucine-rich repeat (LRR) domain of the membrane receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1), but it is unknown how steroid binding at the cell surface activates the cytoplasmic kinase domain of the receptor. A family of somatic embryogenesis receptor kinases (SERKs) has been genetically implicated in mediating early brassinosteroid signaling events. We found a direct and steroid-dependent interaction between the BRI1 and SERK1 LRR domains by analysis of their complex crystal structure at 3.3 angstrom resolution. We show that the SERK1 LRR domain is involved in steroid sensing and, through receptor-co-receptor heteromerization, in the activation of the BRI1 signaling pathway. Our work reveals how known missense mutations in BRI1 and in SERKs modulate brassinosteroid signaling and the targeting mechanism of BRI1 receptor antagonists. PMID:23929946
Santiago, Julia; Henzler, Christine; Hothorn, Michael
Somatic embryogenesis in pumpkin can be induced on auxin-containing medium and also on hormone-free medium containing 1mM ammonium (NH(4)(+)) as the sole source of nitrogen. Growth of NH(4)(+)-induced embryogenic tissue was slow and caused considerable acidification of the culture medium. Small spherical cells with dense cytoplasma formed proembryogenic cell clusters that could not develop into late stage embryos. Buffering of NH(4)(+) medium with 25mM 2-(N-morpholino)-ethane-sulfonic acid enhanced tissue proliferation, but no further differentiation was observed. Later stage embryos developed only after re-supply of nitrogen in form of nitrate or l-glutamine. Effects of nitrogen status and pH of culture media on ammonium assimilation were analyzed by following the activity of glutamine synthetase (GS) in relation to phenylalanine ammonia-lyase (PAL). Increased activity of GS and PAL in NH(4)(+) induced tissue coincided with significantly higher activity of stress-related enzymes superoxide dismutase (SOD) and soluble peroxidase (POD), indicating oxidative stress response of embryogenic tissue to NH(4)(+) as the sole source of nitrogen. In addition, considerable increase was observed in callose accumulation and esterase activity, the early markers of somatic embryogenesis. Activity of stress-related enzymes decreased after the re-supply of nitrate (20mM) or Gln (10mM) in combination with NH(4)(+) (1mM), which subsequently triggered globular embryo development. Together, these results suggest that stress responses, as affected by nitrogen supply, contribute to the regulation of embryogenic competence in pumpkin. PMID:21807439
Mihaljevi?, Snježana; Radi?, Sandra; Bauer, Nataša; Gari?, Rade; Mihaljevi?, Branka; Horvat, Gordana; Leljak-Levani?, Dunja; Jelaska, Sibila
The genetic basis of the regeneration process in cultured immature embryos of rye (Secale cereale L.) was analyzed. The experiments were designed to reveal differences between the in vitro culture responses of two inbred lines: L318 (a high regeneration ability) and L9 (a low potential for regeneration). The rye ortologues of plant genes previously recognized as crucial for somatic embryogenesis and morphogenesis in vitro were identified. Using oligonucleotide primers designed to conserved regions of the genes Somatic Embryogenesis Receptor-like Kinase (SERK), Leafy Cotyledon 1 (LEC1), Viviparous 1 (VP1) and NiR (encoding ferredoxin-nitrite reductase), it was possible to amplify specific homologous sequences from rye RNA by RT-PCR. The transcript levels of these genes were then measured during the in vitro culture of zygotic embryos, and the sites of expression localized. The expression profiles of these genes indicate that their function is likely to be correlated with the in vitro response of rye. In line L9, increased expression of the rye SERK ortologue was observed at most stages during the culture of immature embryos. The suppression of ScSERK expression appears to start after the induction of somatic embryogenesis and lasts up to plant regeneration. The rye ortologues of the LEC1 and VP1 genes may function in a complimentary manner and have a negative effect on the production of the embryogenic callus. The expression of the rye NiR ortologue during in vitro culture reveals its importance in the process of plant regeneration.
Background and Aims In Tilia amurensis, two types of trichomes (hairy and glandular) develop from epidermal surfaces of cotyledons and hypocotyls of zygotic embryos soon after germination. Here, it is demonstrated that glandular trichome initials develop directly into somatic embryos when treated in vitro with 2,4-dichlorophenoxyacetic acid (2,4-D). Methods Zygotic embryos of Tilia amurensis were cultured on Murashige and Skoog medium with 3 % sucrose and various concentrations (0, 2·2, 4·4 and 8·8 µm) of 2,4-D. Morphological development of trichomes and somatic embryos was analysed by scanning electron microscope and light microscope after histological sectioning. Key Results In zygotic embryos cultured on medium with 4·4 µm 2,4-D, formation of hairy trichomes was completely suppressed but formation of glandular trichome initials increased. That some filamentous trichome initials developed directly into somatic embryos was confirmed by histological and scanning electron microscope observation. When explants with different stages of trichome initials (two-, four- and eight-celled filamentous and fully mature trichomes) were temporally pre-treated with 4·4 µm 2,4-D for 24 h and transferred into hormone-free medium, two-celled and four-celled filamentous trichome initials were the effective stage of trichomes for somatic embryo induction. Conclusions It is suggested that early developing filamentous trichome initials have developmental plasticity and that with 2,4-D treatment these trichome initials develop directly into somatic embryos.
Kim, T. D.; Lee, B. S.; Kim, T. S.; Choi, Y. E.
The aim of this study was to improve the direct somatic embryogenesis and initiate embryogenic callus formation in camphor\\u000a tree (Cinnamomum camphora L.) on hormone-free medium. The influence of osmotic stress pretreatment of immature zygotic embryos (0.5 and 1.0 M solution\\u000a of sucrose for 12, 24, 48, 72, 96, 120, and 144 h at 4 or 25°C) before cultured on hormone-free medium,
Xueping Shi; Xigang Dai; Guofeng Liu; Manzhu Bao
With the purpose of increasing the embryogenesis regeneration process in vitroplants obtained from somatic embryos of the indica rice variety CR-5272 (Oryza sativa L.), two independent experiments were performed. The first experiment consisted in the effect of combination of three concentrations of the gelling agent Phytagel (1.8, 2.4, and 3 gL-1) and four 2,4-D concentrations (2.26, 4.52, 6.78, and 9.05
Allan Meneses; Dora Flores; Miguel Muñoz; Griselda Arrieta; Ana M. Espinoza
Summary The most important advantage of cloning conifers by somatic embryogenesis (SE) is that the embryogenic tissue can be cryopreserved\\u000a without changing its genetic make-up and without loss of juvenility. This offers an opportunity to develop high-value clonal\\u000a varieties by defrosting and repropagating cryopreserved clones after genetic testing has shown which clones are the best performers.\\u000a In the current absence of
Y. S. Park; J. D. Barrett; J. M. Bonga
The effect of sugars, gibberellic acid (GA3) and abscisic acid (ABA) on somatic embryogenesis from inter-nodal explant-derived callus of Tylophora indica(Burm. f.) Merrill has been investigated. Embryogenic calli were produced from inter-nodal explants and the best result was achieved using Murashige and Skoog (MS) medium supplemented with 4 ?mol\\/L 2, 4-Dichlorophenoxyacetic acid (2, 4-D). Up to 69% of such embryogenic
T. Dennis Thomas
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. PMID:16972095
Estabrooks, Tammy; Browne, Robin; Dong, Zhongmin
Seedlings of carrot (Daucus carota L. cv. Red Cored Chantenay) formed somatic embryos when cultured on medium containing abscisic acid (ABA) as the sole source of growth regulator. The number of embryos per number of seedlings changed depending on the concentration of ABA added to the medium, with a maximum embryo number at 1 x 10(-4) M ABA. Seedling age was critical for response to exogenous ABA; no seedling with a hypocotyl longer than 3.0 cm was able to form an embryo. Removal of shoot apices from seedlings completely inhibited the embryogenesis induced by application of exogenous ABA, suggesting that the action of ABA requires some substance(s) that is translocated basipetally from shoot apices through hypocotyls. Histologically, somatic embryos shared common epidermal cells and differentiated not through the formation of embryogenic cell clumps, but directly from epidermal cells. These morphological traits are distinct from those of embryogenesis via formation of embryogenic cell clumps, which has been found in embryogenic carrot cultures established using 2,4-dichlorophenoxyacetic acid or other auxins. These results suggest that ABA acts as a signal substance in stress-induced carrot seedling somatic embryogenesis. PMID:11089691
Nishiwaki, M; Fujino, K; Koda, Y; Masuda, K; Kikuta, Y
Influence of plant growth regulators, carbon sources and iron on the cyclic secondary somatic embryogenesis and plant regeneration of transgenic cherry rootstock `Colt' ( Prunus avium × P. pseudocerasus )
The frequency of long-term secondary somatic embryogenesis and shoot meristem development from embryogenic masses of the cherry rootstock `Colt' ( Prunus avium × P. pseudocerasus), differentiated from transgenic roots containing the T-DNA of Agrobacterium rhizogenes, has opened the way for genetic improvement by biotechnological techniques. Whole plants were produced by stimulating shoot meristem development from somatic embryos. The combination of
Patricia Gutièrrez pesce; Eddo Rugini
Summary • Here, embryo-specific patterns of glutamine synthetase (GS) genes were studied for the first time using pine somatic and zygotic embryogenesis as model systems. • GS1a expression was absent in zygotic embryos whereas it was detected in the cotyledons of somatic embryos at late developmental stages along with transcripts for photosynthesis genes and arginase. These findings suggest that germination
Maria J Perez Rodriguez; Maria Fernanda Suarez; Raul Heredia; Concepcion Avila; David Breton; Jean-Francois Trontin; Lada Filonova; Peter Bozhkov; Sara von Arnold; Luc Harvengt; Francisco M. Canovas
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
Maldonado-Borges, Josefina Ines; Ku-Cauich, José Roberto; Escobedo-Graciamedrano, Rosa Maria
It was shown earlier, that ginseng embryogenic cell culture 2c3 was obtained as a result of callus cells transformation with the Agrobacterium rhizogenes rolC oncogene. In the present report we determine that inhibitors of Ca2+-channels (LaCl3, verapamil, niflumic acid) certainly lowered the quantity of somatic embryos in the 2c3 cell culture. This is the evidence of the influence of calcium-dependent signal system on plant embryogenesis. Protein kinases inhibitors W7 and H7 also caused the lowering of somatic embryos quantity in the 2c3 cell culture. We analysed changes of CDPK genes expression in embryogenic 2c3 cell culture. Total expression decreased 1.2-1.5 times comparing with the control callus culture. CDPK expression in the 2c3 embryogenic culture lowered by the inhibition of expression of the gene subfamilies PgCDPK1 (PgCDPK1a and PgCDPK1b) and PgCDPK3 (PgCDPK3a). At the same time, expression of PgCDPK2 gene subfamily (PgCDPK2b and PgCDPK2d) was increased. We suppose that genes of PgCDPK2 subfamily might be responsible for the embryogenesis initiation in the 2c3 ginseng cell culture. It was shown for the first time that the rolC gene and the process of embryogenesis could change expression of particular forms of CDPK genes. PMID:18610836
Kiselev, K V; Gorpenchenko, T Iu; Chernoded, G K; Dubrovina, A S; Grishchenko, O V; Bulgakov, V P; Zhuravlev, Iu N
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.
Maldonado-Borges, Josefina Ines; Ku-Cauich, Jose Roberto; Escobedo-GraciaMedrano, Rosa Maria
Expression of knotted1 ( kn1) and ZmLEC1, a maize homologue of the Arabidopsis LEAFY COTYLEDON1 ( LEC1) was studied using in situ hybridization during in vitro somatic embryogenesis of maize ( Zea mays L.) genotype Hi-II. Expression of kn1 was initially detected in a small group of cells (5-10) in the somatic embryo proper at the globular stage, in a specific region where the shoot meristem is initiating at the scutellar stage, and specifically in the shoot meristem at the coleoptilar stage. Expression of ZmLEC1 was strongly detected in the entire somatic embryo proper at the globular stage, gradually less in the differentiating scutellum at the scutellar and coleoptilar stages. The results of analyses show that the expression pattern of kn1 during in vitro somatic embryogenesis of maize is similar to that of kn1 observed during zygotic embryo development in maize. The expression pattern of ZmLEC1 in maize during in vitro development is similar to that of LEC1 in Arabidopsis during zygotic embryo development. These observations indicate that in vitro somatic embryogenesis likely proceeds through similar developmental pathways as zygotic embryo development, after somatic cells acquire competence to form embryos. In addition, based on the ZmLEC1 expression pattern, we suggest that expression of ZmLEC1 can be used as a reliable molecular marker for detecting early-stage in vitro somatic embryogenesis in maize. PMID:12029467
Zhang, Shibo; Wong, Laurie; Meng, Ling; Lemaux, Peggy G
Of the various alternatives for cloning elite conifers, somatic embryogenesis (SE) appears to be the best option. In recent years, significant areas of lodgepole pine (Pinus contorta) forest have been devastated by the mountain pine beetle (MPB) in Western Canada. In an attempt to establish an SE propagation system for MPB-resistant lodgepole pine, several families displaying varying levels of resistance were selected for experimentation involving shoot bud and immature seed explants. In bud cultures, eight embryogenic lines were induced from 2 of 15 genotypes following various treatments. Genotype had an important influence on embryogenic culture initiation, and this effect was consistent over time. These lines were identified by microscopic observation and genetic markers. Despite the abundance of early somatic embryos, the cultures have yet to develop into mature embryos. In contrast, immature zygotic embryos (ZEs) cultured from megagametophytes initiated SE at an early dominance stage via nodule-type callus in 1 of 10 genotypes. As part of the study, putative embryogenesis-specific genes, WOX2 (WUSCHELL homeobox 2) and HAP3A, were analyzed in cultures of both shoot bud explants and ZEs. On the basis of these analyses, we postulate that PcHAP3A was expressed mainly in callus and may be involved in cell division, whereas WOX2 was expressed mainly in embryonal mass (EM)-like tissues. The findings from this study, based on molecular assessment, suggest that the cell lines derived from bud cultures were truly EM. Moreover, these experimental observations suggest that PcWOX2 could be used as an early genetic marker to discriminate embryogenic cultures from callus. PMID:20935320
Park, So-Young; Klimaszewska, Krystyna; Park, Ji-Young; Mansfield, Shawn D
It has been shown previously that height growth and bud phenology are influenced by the temperature during zygotic embryogenesis in Picea abies. To test whether this phenomenon operates within individual plants, clones produced through somatic embryogenesis were used. Seeds were from a full-sib family produced in both a cold (outdoor) and a warm (inside a glasshouse) environment. Embryogenic clones derived from mature zygotic embryos from both crossing environments were cultured at 18, 23 and 28 degrees C during the proliferation and embryo maturation steps. After the second growing season in a glasshouse, plants from the warm seed production environment were taller and had significantly later bud set. For the first time, it is also shown that plants are influenced by the in vitro temperature during somatic embryo development. The warmer the temperature, the later the plants formed terminal buds. The differences were similar to those produced by a provenance separation of 4-6 degrees of latitude. The results indicate that there exists a mechanism in P. abies that operates during embryo development and adjusts the timing of bud set in accordance with the temperature conditions in which the mother tree lives. This in turn counteracts negative effects of gene flow among populations located along altitudinal and latitudinal gradients. PMID:17924949
Kvaalen, Harald; Johnsen, Oystein
An improved method of direct somatic embryogenesis (SE) was developed in Swertia chirata for the first time using leaves and roots of in vitro-grown young seedlings. In the present study, 2,4-dichlorophenoxyacetic\\u000a acid (2,4-D) was assessed individually and in combination with other auxins, as well as with cytokinin for its effectiveness\\u000a to induce somatic embryos. Leaf explants with abaxial side in
K. Balaraju; S. Saravanan; P. Agastian; S. Ignacimuthu
Embryogenic suspensions of grapevine (Vitis vinifera L.) were initiated from somatic embryos of `Thompson Seedless' and `Chardonnay'. Suspension cultures consisted of proembryonic\\u000a masses (PEM) that proliferated without differentiation in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). `Chardonnay'\\u000a somatic embryos developed fully from PEMs following subculture in medium without 2,4-D; however, somatic embryo development\\u000a did not advance beyond the heart stage in
S. Jayasankar; D. J. Gray; R. E. Litz
Direct somatic embryogenesis was obtained from intact and fragmented zygotic embryos of Myristica malabarica, an endemic, threatened medicinal species of Western Ghats of Southern India while cultured in Murashige and Skoog medium containing activated charcoal. In the absence of activated charcoal there was no embryogenic response but only callus formation in zygotic embryos and their fragments. The addition of gibberellic
R. Indira Iyer; G. Jayaraman; A. Ramesh
Leaf discs from four strains and petioles from six strains of Gossypium hirsutum were cultured on a variety of media. Callus formed from explants on all media, though embryogenesis was highly specific. Embryos formed from only three strain x media combinations. A small percentage of these embryos developed into plantlets. These results demonstrate that cotton plants can be obtained from
Nick J. Gawel; Arelli P. Rao; Carol D. Robacker
The aim of this study was the improvement of embryo production in wheat anther culture. Three butanol alcohols, n-butanol, sec-butanol and tert-butanol, were evaluated for their effect on microspore embryogenesis in two spring cultivars\\u000a of wheat, Pavon and Caramba. Application of n-butanol, at 0.1 and 0.2% (v\\/v) in the induction media for 5 h, highly improved embryo production in both cultivars.
M. Soriano; L. Cistué; A. M. Castillo
Carrot (Daucus carota L.) cells were transformed with Agrobacterium tumefaciens strains containing 3[prime]-truncated mouse ornithine decarboxylase (ODC) cDNA under the control of a cauliflower mosaic virus 35S promoter. A neomycin phosphotransferase gene linked with a nopaline synthase promoter was used to select transformed cell lines on kanamycin. Although the nontransformed cells contained no ODC, high amounts of mouse-specific ODC activity were observed in the transformed cells. Transgenic cells showed a significant increase in the cellular content of putrescine compared to control cells. Spermidine, however, remained unaffected. Not only did the transformed cells exhibit improved somatic embryogenesis in the auxin-free medium, they also regenerated some embryos in the presence of inhibitory concentrations of 2,4-dichlorophenoxyacetic acid. These cells acquired tolerance to [alpha]-difluoromethylarginine (a potent inhibitor of arginine decarboxylase) at concentrations that inhibit growth as well as embryogenesis in nontransformed carrot cells, showing that the mouse ODC can replace the carrot arginine decarboxylase for putrescine biosynthesis in the transgenic cells.
Bastola, D. R.; Minocha, S. C.
Callus was obtained from immature zygotic embryos of semminiferous species (diploids) of Musa sp. using a medium derived from that of Murashige and Skoog. Picloram (7.5 ?M) was added and the medium was solidified with gelrite (2 gl(-1)). Differentiation of the first somatic embryos occurred after transfer of the callus in the presence of 7.5 ?M picloram or 5.3 ?M NAA. Somatic embryos germinated on the medium supplemented with 5.3 ?M NAA. Serial sections of zygotic and somatic embryos showed perfect homology in their structure (epidermis, cotyledonary slit, shoot apex and 3 root primordia). Embryonic callus was characterised by a large quantity of protein storage in the cytoplasm. PMID:24240457
Escalant, J V; Teisson, C
Somatic embryos of a seeded diploid ornamental banana (Musa ornata Roxb.) were obtained from zygotic embryos cultured on semi-solid Murashige and Skoog (MS) (1962) medium with the auxin 2,4-D (0.5, 1, 2 mg/l) and 5% CW. Removal of 2,4-D and transferral to Schenk and Hildebrandt (SH) (1972) salts with CW followed by basal MS led to embryo germination and growth. Plantlet production was obtained using filter paper bridges in liquid half-strength SH medium with 1% sucrose. The remarkable phenotypic fidelity of somatic embryos to that of zygotic embryos and the presence of a haustorium-like outgrowth on the somatic embryos is described. PMID:11538845
Cronauer-Mitra, S S; Krikorian, A D
Somatic embryos of a seeded diploid ornamental banana (Musa ornata Roxb.) were obtained from zygotic embryos cultured on semi-solid Murashige and Skoog (MS) (1962) medium with the auxin 2,4-D (0.5, 1, 2 mg/l) and 5% CW. Removal of 2,4-D and transferral to Schenk and Hildebrandt (SH) (1972) salts with CW followed by basal MS led to embryo germination and growth. Plantlet production was obtained using filter paper bridges in liquid half-strength SH medium with 1% sucrose. The remarkable phenotypic fidelity of somatic embryos to that of zygotic embryos and the presence of a haustorium-like outgrowth on the somatic embryos is described. PMID:24241408
Cronauer-Mitra, S S; Krikorian, A D
Summary Proliferative somatic embryogenesis is a regeneration system suitable for mass propagation and genetic transformation of soybean\\u000a [Glycine max (L.) Merr.]. The objective of this study was to examine genotypic effects on induction and maintenance of proliferative embryogenic\\u000a cultures, and on yield, germination, and conversion of mature somatic embryos. Somatic embryos were induced from eight genotypes\\u000a by explanting 100 immature cotyledons
M. A. Bailey; H. R. Boerma; W. A. Parrott
Globular somatic embryos can be induced from immature cotyledons of soybean (Glycine max L. Merr. cv Jack) placed on high levels of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryos develop from the adaxial side of the cotyledon, whereas the abaxial side evolves into a callus. Using a 9,280-cDNA clone array, we have compared steady-state RNA from the adaxial side from which embryos develop and from the abaxial callus at five time points over the course of the 4 weeks necessary for the development of globular embryos. In a second set of experiments, we have profiled the expression of each clone in the adaxial side during the same period. A total of 495 genes differentially expressed in at least one of these experiments were grouped according to the similarity of their expression profiles using a nonhierarchical clustering algorithm. Our results indicate that the appearance of somatic embryos is preceded by dedifferentiation of the cotyledon during the first 2 weeks on auxin. Changes in mRNA abundance of genes characteristic of oxidative stress and genes indicative of cell division in the adaxial side of the cotyledons suggest that the arrangement of the new cells into organized structures might depend on a genetically controlled balance between cell proliferation and cell death. Our data also suggest that the formation of somatic globular embryos is accompanied by the transcription of storage proteins and the synthesis of gibberellic acid.
Thibaud-Nissen, Francoise; Shealy, Robin T.; Khanna, Anupama; Vodkin, Lila O.
A new culture medium, originally designed and shown to grow cell suspensions from a variety of loblolly pine (Pinus taeda L.) explants, was used to study growth and somatic embryogenesis of the wild carrot (Daucus carota L.) in cell suspensions. The new loblolly pine medium (LM) differed from the standard wild carrot medium (WCM) in having very low Ca(2+), very high Mg(2+), and enrichment with PO inf4 (sup3-) and microelements. When WCM was altered to contain levels of Ca(2+) or Ca(2+) and Mg(2+) equivalent to LM, it supported neither growth nor embryogenesis of the wild carrot. However, growth and embryogenesis in LM was superior to WCM. The phosphate level in WCM was found to be suboptimal. PMID:24254074
Litvay, J D; Verma, D C; Johnson, M A
Root explants excised from carnation plants maintained in vitro formed off-white, friable calluses after three weeks of culture\\u000a on Murashige and Skoog (MS) medium supplemented with 1 mg l?1 thidiazuron (TDZ) and 1 mg l?1 ?-naphthalaneacetic acid (NAA). These calluses were subsequently transferred to MS basal medium where, after an additional\\u000a four weeks of culture, approximately 50% of the calluses formed somatic embryos. However, calluses formed
Jinwook Seo; Suk Weon Kim; Sung Ran Min; Jang R. Liu
Leaf segments excised from Centella asiatica, a medicinal and neutraceutical plant, produced abundant somaticembryoswhen cultured onMS mediumwith 9.29 µMkinetin in combination with 2.26 µM2,4-D. Granular, white,shiny clusters of callus developed after 1 week of culture, and then formed heart and cotyledonary stage embryoson the same medium after 4 weeks. Somatic embryos matured and germinated in the presence of MS mediumcontaining
Ch. Paramageetham; G. Prasad Babu; J. V. S. Rao
Plant regeneration in Chlorophytum borivilianum Sant. et Fernand. from embryogenic callus and cell suspension culture and assessment of genetic fidelity of plants derived through somatic embryogenesis.
Efficient in vitro propagation of medicinally important endangered plant C. borivilianum has been achieved through somatic embryogenesis. Solid embryogenic medium [Murashige and Skoog medium containing 1.79 mM NH4NO3, 10.72 mM KNO3, 1.13 ?M 2,4-dichlorophenoxyacetic acid, 7.38 ?M 2-isopentenyladenine and 0.76 mM proline] supplemented with polyethylene glycol and sucrose (3 % each), exhibited 1.88-fold increase in embryo maturation compared to embryogenic medium containing 3 % sucrose. Liquid embryogenic medium supported better somatic embryo production and maturation. Highest total (79) and mature (cotyledonary stage) somatic embryos (38) as well as highest germination (57.5 %) was observed at inoculum density of 0.4 g/40 ml of liquid medium. 5.86 pH level exhibited optimal growth, maturation and germination of somatic embryos. Random amplified polymorphic DNA (RAPD) analysis of C. borivilianum plants regenerated through somatic embryogenesis revealed that they were genetically similar to the mother plant. The protocol established in the present study can be used for rapid mass multiplication of C. borivilianum in bioreactor employing liquid medium. PMID:23814440
Rizvi, Mohd Zahid; Kukreja, Arun Kumar; Bisht, Narendra Singh
By flow cytometric experiments in Cyclamen persicum both with propidium iodide (PI) and DAPI (4?,6-Diamidino-2-phenylindol)-staining we were able to present (1) a new estimation\\u000a for the absolute DNA content in the range of 3.17 pg DNA\\/2C and (2) ploidy abnormalities which were detected in the pathway\\u000a of somatic embryogenesis. These aberrations might have arisen from ploidy mutations or abnormal polyploidization processes.
Thomas Borchert; Jörg Fuchs; Traud Winkelmann; Annette Hohe
The usefulness of random amplified polymorphic DNA (RAPD) in assessing the genetic stability of somatic embryogenesis-derived populations of black spruce [Picea mariana (Mill.) B.S.P.] was evaluated. Three arbitrary 11-mer primers were successfully used to amplify DNA from both in-vivo and in-vitro material. Twenty-five embryogenic cell lines, additional zygotic embryos and megagametophytes from three controlled crosses involving four selected genotypes of
N. Isabel; L. Tremblay; M. Michaud; F. M. Tremblay; J. Bousquet
This study describes culture conditions for a plant regeneration system via a combined pathway of somatic embryogenesis and\\u000a organogenesis in root explant cultures of the commercial rose cultivar 'Charming'. Root explants formed white calluses at\\u000a a frequency of 30% after 6 weeks of culture on Schenk and Hildebrandt (SH) medium supplemented with 11 mg l?1 2,4-dichlorophenoxyacetic acid. After 6 weeks of transfer to SH
Suk Weon Kim; Myung Jin Oh; Jang R. Liu
Somatic embryo formation was induced from cotyledon explants of Styrian pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) by using a solid MS medium supplemented with 16.11?µM NAA and 4.44?µM BA or 26.85?µM NAA and 13.32?µM BA. The callus proliferation was more efficient on medium supplemented with 26.85?µM NAA and 13.32?µM BA. In contrast, the embryogenic response was higher
A. Urbanek; B. Zechmann; M. Müller
We report the isolation and regeneration of protoplasts from an embryogenic banana (Musa spp.) cell suspension culture initiated from in vitro proliferating meristems. A high yielding isolation method (up to 6×10(7) protoplasts.ml(-1) packed cells) is discussed. Optimal regeneration, with more than 50% of the protoplasts showing initial cell division, occurred when high inoculation densities (10(6) protoplasts.ml(-1)) or nurse cultures were applied. Under these conditions, the frequency of microcolony formation was 20-40%. These microcolonies developed directly, without an intervening callus phase, into somatic embryos which later germinated and formed plantlets. PMID:24197341
Panis, B; Van Wauwe, A; Swennen, R
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.
Couillerot, Jean-Paul; Windels, David; Vazquez, Franck; Michalski, Jean-Claude; Hilbert, Jean-Louis; Blervacq, Anne-Sophie
To get a broader view on the molecular mechanisms underlying somatic embryogenesis (SE) in cotton (Gossypium hirsutum L.), global analysis of cotton transcriptome dynamics during SE in different sister lines was performed using RNA-Seq. A total of 204?349 unigenes were detected by de novo assembly of the 214?977?462 Illumina reads. The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) measurements were positively correlated with the RNA-Seq results for almost all the tested genes (R(2) ?=?0.841, correlation was significant at the 0.01 level). Different phytohormone (auxin and cytokinin) concentration ratios in medium and the endogenous content changes of these two phytohormones at two stages in different sister lines suggested the roles of auxin and cytokinin during cotton SE. On the basis of global gene regulation of phytohormone-related genes, numerous genes from all the differentially expressed transcripts were involved in auxin and cytokinin biosynthesis and signal transduction pathways. Analyses of differentially expressed genes that were involved in these pathways revealed the substantial changes in gene type and abundance between two sister lines. Isolation, cloning and silencing/overexpressing the genes that revealed remarkable up- or down-expression during cotton SE were important. Furthermore, auxin and cytokinin play a primary role in SE, but potential cross-talk with each other or other factors remains unclear. PMID:23710882
Xu, Zhenzhen; Zhang, Chaojun; Zhang, Xueyan; Liu, Chuanliang; Wu, Zhixia; Yang, Zuoren; Zhou, Kehai; Yang, Xiaojie; Li, Fuguang
Background To date, several studies have indicated a major role for microRNAs (miRNAs) in regulating plant development, but miRNA-mediated regulation of the developing somatic embryo is poorly understood, especially during early stages of somatic embryogenesis in hardwood plants. In this study, Solexa sequencing and miRNA microfluidic chips were used to discover conserved and species-specific miRNAs during somatic embryogenesis of hybrid yellow poplar (Liriodendron tulipifera×L. chinense). Methodology/Principal Findings A total of 17,214,153 reads representing 7,421,623 distinct sequences were obtained from a short RNA library generated from small RNAs extracted from all stages of somatic embryos. Through a combination of deep sequencing and bioinformatic analyses, we discovered 83 sequences with perfect matches to known miRNAs from 33 conserved miRNA families and 273 species-specific candidate miRNAs. MicroRNA microarray results demonstrated that many conserved and species-specific miRNAs were expressed in hybrid yellow poplar embryos. In addition, the microarray also detected another 149 potential miRNAs, belonging to 29 conserved families, which were not discovered by deep sequencing analysis. The biological processes and molecular functions of the targets of these miRNAs were predicted by carrying out BLAST search against Arabidopsis thaliana GenBank sequences and then analyzing the results with Gene Ontology. Conclusions Solexa sequencing and microarray hybridization were used to discover 232 candidate conserved miRNAs from 61 miRNA families and 273 candidate species-specific miRNAs in hybrid yellow poplar. In these predicted miRNAs, 64 conserved miRNAs and 177 species-specific miRNAs were detected by both sequencing and microarray hybridization. Our results suggest that miRNAs have wide-ranging characteristics and important roles during all stages of somatic embryogenesis in this economically important species.
Qiu, Shuai; Zhang, Yanjuan; Wang, Pengkai; Yang, Liwei; Lu, Ye; Shi, Jisen
Procedures were developed for disinfestation of non-dormant basal plate tissue excised from field grown basal plate tissue of diploid Allium fistulosum × A. cepa F1 hybrid onions. Contamination levels varied with the season and vegetative development of plant material. Callus initiated from basal plate tissue and immature inflorescences of the F1 hybrids was maintained on a BDS-based medium containing 0.75 mg/l picloram and 2.0 mg/l BA. When this medium was supplemented with vitamins and glycine, and with proline at 2.5 gm/1, somatic embryos began to form. Their development continued on a BDS-based shoot promotion medium containing 0.03 mg/l picloram and 0.32 mg/l 2iP supplemented with vitamins, glycine and proline. Genotypes differed significantly in the numbers of structures regenerated. Plantlets from somatic embryos were rooted into BDS or half-strength BDS medium without growth substances and were successfully transferred to sterilized potting mix in plastic commercial corsage boxes. PMID:24240465
Lu, C C; Currah, L; Peffley, E B
Mature leaf explant derived callus of Tylophora indica (Burm. f.) Merrill yielded somatic embryos on MS medium supplied with BA(1-2 mg/L) or kinetin(1-5 mg/L) or kinetin/BA (1-2 mg/L) used along with IAA(0.1-1 mg/L). Maximum somatic embryos (30) could be recovered from 100 mg of embryogenic callus within 60 days at an optimum concentration of 2 mg/L of BA which was also best suited for providing the maximum conversion rate (90%) of embryoids to plantlets. Kinetin (1-5 mg/L), used as the sole growth hormone, induced the development of embryoids showing either shoot or root primordia in 30% of the cultures. However, embryoids with shoot primordia developed roots upon transfer to medium containing IAA(0.1 mg/L) and kinetin(2 mg/L). Embryoids from all cultures germinated in the initiation medium and were transplanted to sterile vermiculite for hardening. After two weeks of hardening, the plantlets were transferred to the green house where they grew and established well showing a high rate of survival (90%). PMID:11324164
Manjula, S; Job, A; Nair, G M
The EMB-1 mRNA of carrot (Daucus carota) was isolated as an embryo abundant cDNA clone (T.H. Ulrich, E.S. Wurtele, B.J. Nikolau  Nucleic Acids Res 18: 2826). Northern analyses of RNA isolated from embryos, cultured cells, and a variety of vegetative organs indicate that the EMB-1 mRNA specifically accumulates in embryos, beginning at the early stages of embryo development. In situ hybridization with both zygotic and somatic embryos show that the EMB-1 mRNA begins to accumulate at low levels throughout globular embryos. Accumulation of EMB-1 mRNA increases and becomes more localized as embryos mature; in torpedo embryos, EMB-1 mRNA preferentially accumulates in the meristematic regions, particularly the procambium. The similarity in distribution of EMB-1 mRNA in both zygotic and somatic embryos indicates that much of the spatial pattern of expression of the emb-1 gene is dependent on the developmental program of the carrot embryo and does not require maternal or endosperm factors. The EMB-1 protein (relative molecular weight 9910) is a very hydrophilic protein that is a member of a class of highly conserved proteins (typified also by the Em protein of wheat and the Lea D19 protein of cotton) that may be ubiquitous among angiosperm embryos but whose functions are as yet unknown. The carrot genome appears to contain one or two copies of the emb-1 gene. A 1313-base pair DNA fragment of the carrot genome containing the emb-1 gene was isolated and sequenced. The gene is interrupted by a single intron of 99 base pairs. Primer extension experiments identify two EMB-1 mRNAs, differing by 6 bases at their 5' ends that are transcribed from this gene.
Wurtele, E S; Wang, H; Durgerian, S; Nikolau, B J; Ulrich, T H
Different NAA plus kinetin or BA combinations were tested on Francia Pernod fennel seedlings for callus induction and plant\\u000a regeneration. Callogenesis from hypocotyls was obtained in all auxin\\/cytokinin-containing media. The organogenic response\\u000a was observed especially in presence of NAA plus kinetin. The highest frequency of shoot regeneration was found when the auxin\\u000a and kinetin were used at a 1:1 ratio.
M. Anzidei; A. Bennici; S. Schiff; C. Tani; B. Mori
SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) genes have been demonstrated to play a role in somatic embryogenesis in several plant species. As more is learnt about these genes, the view of their role in plant development has broadened. The Medicago truncatula MtSERK1 gene has been associated with somatic embryogenesis and in vitro root formation. In order to study the role of MtSERK1 in development further, the MtSERK1 promoter sequence has been isolated and cloned into a promoter–GUS analysis vector. SERK1 promoter-driven GUS expression was studied in A. tumefaciens-transformed cultures and regenerated plants, in A. rhizogenes-transformed root clones, and in nodulation. In embryogenic cultures, GUS staining is detected after 2 d of culture at the edge of the explant and around vascular tissue. Expression at the explant edge intensifies over subsequent days and then is lost from the edge as callus formation moves inward. MtSERK1 expression appears to be associated with new callus formation. When somatic embryos form, GUS staining occurs throughout embryo development. Zygotic embryos show expression until the heart stage. The in planta studies reveal a number of interesting expression patterns. There appear to be three types. (i) Expression associated with the primary meristems of the root and shoot and the newly formed meristems of the lateral roots and nodule. (ii) Expression at the junction between one type of tissue or organ and another. (iii) Expression associated with the vascular tissue procambial cells. The data led us to conclude that MtSERK1 expression is associated with developmental change, possibly reflecting cellular reprogramming.
Nolan, Kim E.; Kurdyukov, Sergey; Rose, Ray J.
An alternative in vitro protocol for embryo induction directly from intact living seedlings of Phalaenopsis aphrodite subspecies formosana was established in this study. Without the supplementation of plant growth regulators (PGRs), no embryos were obtained from all the seedlings when cultured on the solid medium. In contrast, embryos formed from the seedlings on the 2-layer medium and the 2-step culture system without the use of PGRs. It was found that the age of the seedlings affected embryo induction. The 2-month-old seedlings typically had higher embryogenic responses when compared with the 4-month-old seedlings in the 2-layer medium or 2-step system. For the 2-month-old seedlings, 1?mg/L TDZ resulted in the highest number of embryos at the distal site of the shoot. However, on the leaves' surface, 0.5?mg/L TDZ induced the highest number of embryos. When the 2-month-old seedlings were cultured using the 2-step method at 1?mg/L of TDZ, the highest embryogenic response was obtained, with an average of 44 embryos formed on each seedling. These adventitious embryos were able to convert into plantlets in a PGR-free 1/2 MS medium, and the plantlets had normal morphology and growth.
The LEAFY COTYLEDON2 (LEC2) transcription factor with a plant-specific B3 domain plays a central role in zygotic and somatic embryogenesis (SE). LEC2 overexpression induced in planta leads to spontaneous somatic embryo formation, but impairs the embryogenic response of explants cultured in vitro under auxin treatment. The auxin-related functions of LEC2 appear during SE induction, and the aim of the present study was to gain further insights into this phenomenon. To this end, the effect of LEC2 overexpression on the morphogenic responses of Arabidopsis explants cultured in vitro under different auxin treatments was evaluated. The expression profiles of the auxin biosynthesis genes were analysed in embryogenic cultures with respect to LEC2 activity. The results showed that LEC2 overexpression severely modifies the requirement of cultured explants for an exogenous auxin concentration at a level that is effective in SE induction and suggested an increase in the auxin content in 35S::LEC2-GR transgenic explants. The assumption of an LEC2 promoted increase in endogenous auxin in cultured explants was further supported by the expression profiling of the genes involved in auxin biosynthesis. The analysis indicated that YUCCAs and TAA1, working in the IPA-YUC auxin biosynthesis pathway, are associated with SE induction, and that the expression of three YUCCA genes (YUC1, YUC4 and YUC10) is associated with LEC2 activity. The results also suggest that the IAOx-mediated auxin biosynthesis pathway involving ATR1/MYB34 and CYP79B2 does not seem to be involved in SE induction. We conclude that de novo auxin production via the tryptophan-dependent IPA-YUC auxin biosynthesis pathway is implicated in SE induction, and that LEC2 plays a key role in this mechanism. PMID:23722561
Wójcikowska, Barbara; Jaskó?a, Karolina; G?siorek, Przemys?aw; Meus, Magdalena; Nowak, Katarzyna; Gaj, Ma?gorzata D
Two cell lines of birch (Betula pendula Roth.), one potentially embryogenic given the right inductive conditions and one which\\u000a never has shown any embryogenic capacity, were both subjected to conditions inductive and non-inductive for somatic embryogenesis.\\u000a Cells from these treatments were harvested at intervals over a 3-week period and washed with salt solution to wash off proteins\\u000a loosely attached to
A. K. Hvoslef-Eide; F. M. K. Corke
An efficient plant regeneration protocol through indirect somatic embryogenesis pathway via callus had been developed from the leaf explant of an ornamental bulbaceous plant Drimiopsis kirkii. Optimum friable calli were induced on Murashige and Skoog (MS) basal medium supplemented with 3.0 mg/l of 2,4-dichlorophenoxyacetic acid and 1.0 mg/l of ?-naphthalene acetic acid (NAA). On subculturing the callus on MS medium supplemented with 2.5 mg/l of thidiazuron (TDZ), 73.3 % of the cultures responded with 20.4?±?0.3 somatic embryos (SEs) per 500 mg callus at different stages of development after 6 weeks of culture. The highest response of 86.7 % with 28.3?±?0.5 embryos per 500 mg callus was observed on MS medium supplemented with 2.5 mg/l TDZ and 1.0 mg/l NAA. SEs were encapsulated in calcium alginate beads for the production of synthetic seeds (SSs) and their storability was investigated. The highest SS germination (93.3 %) was observed in 1.0 % sodium alginate followed by 86.7 % germination with 2.5 % sodium alginate. The SSs were stored at three different temperatures (4, 15, and 24?ºC) up to 6 months. The SSs kept at 15 °C showed 64.4 % germinability even after 4 months of storage. Both nonencapsulated and encapsulated SE-derived plants were successfully transferred to soil with 93.3 and 88.3 % survival rate accordingly. Randomly amplified polymorphic DNA (RAPD) analysis revealed that there were no somaclonal variations among the plants produced via somatic embryogenesis and they are true-to-type to their parental plant. These results confirmed the most reliable methods, which can be further used for genetic transformation studies as well as for mass propagation of ornamental D. kirkii at a commercial level. PMID:24604129
Haque, Sk Moquammel; Ghosh, Biswajit
Somatic embryogenesis (SE) is the process by which cells become dedifferentiated and reprogram to follow an embryogenic pathway. It is important for regeneration of transgenic plants as well as for propagation of certain genotypes. However, competence for SE varies, even among genotypes of a species, and the basis for this variation is not understood. We have found that the MADS-box transcription factor (Glycine max) AGAMOUS-Like 15 [(Gm)AGL15] promotes SE in Arabidopsis and in soybean when overexpressed. In soybean, part of the promotion of SE is via GmAGL15-mediated control of ethylene biosynthesis and response. Addition of ACC, the precursor to ethylene, to culture media enhanced SE in Arabidopsis and soybean. Transcription factors important for embryogenesis responded directly to GmAGL15 and to ethylene accumulation. Here we correlate ethylene production and patterns of gene expression with SE potential of soybean genotypes. However, other results indicate that there is not a complete positive correlation between ethylene production and SE, indicating that the interactions between hormones, gene expression and developmental outcomes are complex.
Zheng, Qiaolin; Zheng, Yumei; Perry, Sharyn E
Somatic embryogenesis (SE) is the process by which cells become dedifferentiated and reprogram to follow an embryogenic pathway. It is important for regeneration of transgenic plants as well as for propagation of certain genotypes. However, competence for SE varies, even among genotypes of a species, and the basis for this variation is not understood. We have found that the MADS-box transcription factor (Glycine max) AGAMOUS-Like 15 [(Gm)AGL15] promotes SE in Arabidopsis and in soybean when overexpressed. In soybean, part of the promotion of SE is via GmAGL15-mediated control of ethylene biosynthesis and response. Addition of ACC, the precursor to ethylene, to culture media enhanced SE in Arabidopsis and soybean. Transcription factors important for embryogenesis responded directly to GmAGL15 and to ethylene accumulation. Here we correlate ethylene production and patterns of gene expression with SE potential of soybean genotypes. However, other results indicate that there is not a complete positive correlation between ethylene production and SE, indicating that the interactions between hormones, gene expression and developmental outcomes are complex. PMID:23838957
Zheng, Qiaolin; Zheng, Yumei; Perry, Sharyn E
Somatic embryogenesis (SE), which resembles zygotic embryogenesis, is an essential component of the process of plant cell differentiation and embryo development. Although microRNAs (miRNAs) are important regulators of many plant develop- mental processes, their roles in SE have not been thoroughly investigated. In this study, we used deep-sequencing, computational, and qPCR methods to identify, profile, and describe conserved and novel miRNAs involved in longan (Dimocarpus longan) SE. A total of 643 conserved and 29 novel miRNAs (including star strands) from more than 169 miRNA families were identified in longan embryogenic tissue using Solexa sequencing. By combining computational and degradome sequencing approaches, we were able to predict 2063 targets of 272 miRNAs and verify 862 targets of 181 miRNAs. Target annotation revealed that the putative targets were involved in a broad variety of biological processes, including plant metabolism, signal transduction, and stimulus response. Analysis of stage- and tissue-specific expressions of 20 conserved and 4 novel miRNAs indicated their possible roles in longan SE. These miRNAs were dlo-miR156 family members and dlo-miR166c* associated with early embryonic culture developmental stages; dlo-miR26, dlo-miR160a, and families dlo-miR159, dlo-miR390, and dlo-miR398b related to heart-shaped and torpedo- shaped embryo formation; dlo-miR4a, dlo-miR24, dlo-miR167a, dlo-miR168a*, dlo-miR397a, dlo-miR398b.1, dlo-miR398b.2, dlo-miR808 and dlo-miR5077 involved in cotyledonary embryonic development; and dlo-miR17 and dlo-miR2089*-1 that have regulatory roles during longan SE. In addition, dlo-miR167a, dlo-miR808, and dlo-miR5077 may be required for mature embryo formation. This study is the first reported investigation of longan SE involving large-scale cloning, characterization, and expression profiling of miRNAs and their targets. The reported results contribute to our knowledge of somatic embryo miRNAs and provide insights into miRNA biogenesis and expression in plant somatic embryo development.
Lin, Yuling; Lai, Zhongxiong
This study established a method of regenerating Spathiphyllum 'Supreme' through direct somatic embryo- genesis. Somatic embryos occurred in leaf and petiole explants cultured in the dark on a Murashige and Skoog basal medium supplemented with 2.27, 4.54, or 9.08 lM N-phenyl-N 0 -1,2,3-thiadiazol-5-ylurea (TDZ) in combina- tion with 1.08 lM a-naphthalene acetic acid or 2.26 lM 2,4-dichlorophenoxyacetic acid (2,4-D). Explants
Jietang ZhaoJin; CuiJuanxu Liu; J. HennyJianjun Chen
The grapevine genetic transformation programs have relayed on the use of solid media-based somatic embryogenesis. To reach a high throughput of candidate gene evaluation in 'Thompson Seedless', a semi-automatic system allowing viable transformation of explants was designed. An intermediate procedure using liquid media and agitated flasks was first characterized, leading to reduction in the biomass duplication time of pro-embryogenic (PE) cells from 30 d in dishes to 14 d. The oxygen transfer coefficient value in this system was 213h(-1) at 120rpm and 25 degrees C with a 16/8-h (light/darkness) photoperiod. The scaling-up to the air-lift bioreactor decreased the biomass duplication time of PE cells up to 5.3 d post-inoculation (pi) and an average volumetric productivity of 1.6g/(dxL). Although slight browning was seen in the explants during the phase of 8-14 d pi, no losses in their viability and regenerative capability were observed. Cultured cells showed normal elongation in the transition from heart- to the torpedo-shape and finally to advanced developmental stages, with radicle emergence and whole plant generation. Agrobacterium-mediated transformation of cells was efficiently incorporated after this multiplication process by use of conventional procedures in dishes, allowing the generation of transgenic plantlets confirmed by PCR. PMID:18984020
Tapia, Eduardo; Sequeida, Alvaro; Castro, Alvaro; Montes, Christian; Zamora, Pablo; López, Reinaldo; Acevedo, Fernando; Prieto, Humberto
Somatic embryogenesis (SE) can be readily induced in leaf explants of the Jemalong 2HA genotype of the model legume Medicago truncatula by auxin and cytokinin, but rarely in wild-type Jemalong. Gibberellic acid (GA), a hormone not included in the medium, appears to act in Arabidopsis as a repressor of the embryonic state such that low ABA (abscisic acid): GA ratios will inhibit SE. It was important to evaluate the GA effect in M. truncatula in order to formulate generic SE mechanisms, given the Arabidopsis information. It was surprising to find that low ABA:GA ratios in M. truncatula acted synergistically to stimulate SE. The unusual synergism between GA and ABA in inducing SE has utility in improving SE for regeneration and transformation in M. truncatula. Expression of genes previously shown to be important in M. truncatula SE was not increased. In investigating genes previously studied in GA investigations of Arabidopsis SE, there was increased expression of GA2ox and decreased expression of PICKLE, a negative regulator of SE in Arabidopsis. We suggest that in M. truncatula there are different ABA:GA ratios required for down-regulating the PICKLE gene, a repressor of the embryonic state. In M. truncatula it is a low ABA:GA ratio while in Arabidopsis it is a high ABA:GA ratio. In different species the expression of key genes is probably related to differences in how the hormone networks optimise their expression.
Nolan, Kim E.; Song, Youhong; Liao, Siyang; Saeed, Nasir A.; Zhang, Xiyi; Rose, Ray J.
Some walnut trees (Juglans regia L.) originating from central Asia display an early flowering phenotype. These "Early Mature" (EM) trees may produce flowers within months of germination. Secondary flowering waves are also observed within a growing season. Inflorescences may carry male, female and hermaphrodite flowers. Progeny obtained from selected EM trees were cultured in vitro to initiate clonal propagation of these genotypes. Embryogenic lines were established through the culture of immature zygotic embryos. Microshoot lines were obtained from germinated somatic or zygotic embryos. Plants showing EM phenotypes were recovered through direct conversion of somatic embryos or adventitious rooting of microcuttings. During the in vitro propagation phase, flower buds were observed on microshoots after three to six subcultures. Histological analysis showed that most of these flowers were hermaphrodite. In vitro apical buds were used to clone the walnut orthologous cDNAs of the AGAMOUS and APETALA 3 MADS-box genes. Northern blots revealed a preferential expression of both of these homeotic genes in flowers. The results highlight the usefulness of EM lines to study the genetic cues controlling flowering and sexual maturity in woody perennials. PMID:14757582
Breton, Christian; Cornu, Daniel; Chriqui, Dominique; Sauvanet, Annie; Capelli, Pierrette; Germain, Eric; Jay-Allemand, Christian
Somatic coliphages have been proposed as indicators of water quality. But several factors have been considered a drawback for their use as indicators. We evaluated the contribution of temperate phages to the numbers of somatic coliphages detected in water by ISO (International Standards Organization) standardised methods. Prophage induction from naturally occurring bacteria was assayed with mitomycin C, ciprofloxacin and UV irradiation. Results indicate that the presence of prophages will not influence the determinations of somatic coliphages in water. PMID:17343678
Muniesa, Maite; Jofre, Juan
A method for the induction of somatic embryos in soybean tissue cultures is described. Cotyledons from immature embryos were utilized as explant source. Supplementing the culture medium with auxins (2,4-D, MCPA, 2,4,5-T, NAA, IAA, IBA) caused formation of meristematic tissue on cotyledon explants. The extent of meristematic tissue formed depended on the kind and concentration of auxin in the culture
Bärbel Lippmann; Gunter Lippmann
Summary Embryogenic callus was induced from explanted pinnae of newly emerged leaves of mature plants ofCeratozamia mexicana var. Robusta (Gymnospermae, Cycadales) on a modified B5 formulation with 1 mg·liter?1 kinetin and 1 mg·liter?1 2,4-dichlorophenoxyacetic acid. Proembryos developed on induction medium, but they were more numerous after subculture onto\\u000a phytohormone-free medium, which also enabled suspensors to elongate. For nearly 1.5 yr after
V. M. Chavez; R. E. Litz; P. A. Moon; K. Norstog
The endosperm plays an important role for the development of zygotic embryos, while somatic embryos lack a seed coat and endosperm and often show physiological disorders. This study aims at elucidating the cellular and physiological processes within the endosperm of the ornamental species Cyclamen persicum Mill. Histological analyses were performed from 0 to 11 weeks after pollination (WAP). At 3WAP, a syncytium was clearly visible with a globular zygotic embryo. From 4WAP, cellularization of the endosperm, at 5WAP a small torpedo shaped embryo, and from 7WAP cell expansion was observed. By 11WAP the endosperm appeared fully differentiated. Total soluble proteins were extracted from the endosperm at 4, 5, 7, 9 and 11WAP and resolved using two dimensional isoelectric focussing/sodium dodecyl sulphate-polyacrylamide gel electrophoresis (2D IEF/SDS-PAGE). A shift from high-molecular-mass proteins to low-molecular-mass proteins during endosperm development was observed. A total of 1137proteinspots/gel were detected in the three protein fractions extracted at 7, 9 and 11WAP. Mass spectrometry analysis of the 48 predominant protein spots in endosperm at 7, 9 and 11WAP resulted in the identification of 62 proteins, ten of which were described for the first time in Cyclamen. Additionally, 186 proteins were identified using the C. persicum embryo proteome reference map. Proteins involved in abscisic acid signalling and oxidative stress responsive proteins were found to be important for seed development in Cyclamen. The new insights into endosperm physiology including storage compounds are discussed. PMID:23352402
Mwangi, Jenniffer Wamaitha; Rode, Christina; Colditz, Frank; Haase, Christin; Braun, Hans-Peter; Winkelmann, Traud
8 halogenated aliphatic hydrocarbons were assayed for their ability to induce somatic segregation in the mould Aspergillus nidulans. Induction of haploidization, mitotic non-disjunction and mitotic crossing-over was studied in heterozygous colonies exposed to the tested chemicals through the detection and phenotypic analysis of segregated sectors. The results obtained show that 1,2-dibromoethane induced all kinds of segregated sectors; 1,2-dichloroethane, allyl chloride, 2-chloroethanol, 2,2-dichloroethanol and 2,2-dichloroacetaldehyde significantly increased the frequency of haploid sectors and diploid non-disjunctional sectors; chloroform and 1,2-dichloropropane were ineffective. PMID:6387478
Crebelli, R; Conti, G; Conti, L; Carere, A
Automation could enhance the use of somatic embryogenesis for micropropagation in two ways: as effective tools for research\\u000a on somatic embryogenesis, and for improving the efficiency of embryo production by reducing labor costs. Processes expected\\u000a to be automated for somatic embryo production are: (1) evaluation of embryogenic cultures, (2) embryo development, (3) harvesting,\\u000a (4) post-harvesting (pre-delivery) processes for enhancing conversion
Yasuomi Ibaraki; Kenji Kurata
High-frequency somatic embryogenesis and shoot regeneration of broccoli (Brassica oleracea var. italica) were achieved. Cotyledon and hypocotyl explants from four varieties of broccoli were cultured on MS and modified MS media (mMS, supplemented with PG-96 organic components) with different combinations of growth regulator. The effects of genotypes, different explants, growth regulator combinations, organic components and AgNO3 on induction of calli
Ying Qin; Hong-Ling Li; Yang-Dong Guo
Isolated plant microspores, when stressed and cultured in vitro, can be diverted from their normal gametophytic pathway towards\\u000a sporophytic development, with the formation of haploid embryos and ultimately doubled-haploid plants. This process is called\\u000a androgenesis or microspore embryogenesis, and is widely used in plant breeding programmes to generate homozygous lines for\\u000a breeding purposes. Protocols for the induction of microspore embryogenesis
Julia Hosp; Simone Faria de Maraschin; Alisher Touraev; Kim Boutilier
In plant embryogenesis, inductive interactions mediated by diffusable signal molecules are most likely of great importance. Evidence has been presented that at late globular stages in plant embryogenesis, perturbation of the polar auxin transport results in abberrant embryo morphology. Rhizobium lipooligosaccharides or Nod factors are a newly discovered class of bacterial molecules that are able to trigger initial steps in
Ed D. L. Schmidt; Anke J. Jong; Sacco C. Vries
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. PMID:24078158
Delporte, Fabienne; Muhovski, Yordan; Pretova, Anna; Watillon, Bernard
The cell line designated PIR-2 is a nonimmunogenic X-ray-induced thymoma of C57BL/6 origin that is unable to induce antitumor immunity in syngeneic lymphocytes in vitro and in mice in vivo. Fusion of PIR-2 with an allogeneic universal fuser A9HT (clone 3c) resulted in the establishment of a somatic cell hybrid designated A9/PIR. C57BL/6 lymphocytes sensitized in vitro with A9/PIR could lyse parental PIR-2 cells, as well as other syngeneic tumors. However, immunization of mice with the hybrid significantly enhanced PIR-2 tumor takes while it partially protected the animals against a challenge with unrelated syngeneic tumors. The results imply that somatic cell hybridization can increase the immunogenicity of an otherwise nonimmunogenic tumor. However, in view of the enhancing effects of hybrid preimmunization on parental tumor cell growth, the possible application of this approach for immunotherapy is questionable.
Yefenof, E.; Goldapfel, M.; Ber, R.
Cancer cells arise through sequential acquisition of mutations in tumor suppressors and oncogenes. c-Jun, a critical component of the AP-1 complex, is frequently overexpressed in diverse tumor types and has been implicated in promoting cellular proliferation, migration, and angiogenesis. Functional analysis of candidate genetic targets using germ line deletion in murine models can be compromised through compensatory mechanisms. As germ line deletion of c-jun induces embryonic lethality, somatic deletion of the c-jun gene was conducted using floxed c-jun (c-junf/f) conditional knockout mice. c-jun-deleted cells showed increased cellular adhesion, stress fiber formation, and reduced cellular migration. The reduced migratory velocity and migratory directionality was rescued by either c-Jun reintroduction or addition of secreted factors from wild-type cells. An unbiased analysis of cytokines and growth factors, differentially expressed and showing loss of secretion upon c-jun deletion, identified stem cell factor (SCF) as a c-Jun target gene. Immunoneutralizing antibody to SCF reduced migration of wild-type cells. SCF addition rescued the defect in cellular adhesion, cellular velocity, directional migration, transwell migration, and cellular invasion of c-jun?/? cells. c-Jun induced SCF protein, mRNA, and promoter activity. Induction of the SCF promoter required the c-Jun DNA-binding domain. c-Jun bound to the SCF promoter in chromatin immunoprecipitation assays. Mutation of the c-Jun binding site abolished c-Jun-mediated induction of the SCF promoter. These studies demonstrate an essential role of c-Jun in cellular migration through induction of SCF.
Katiyar, Sanjay; Jiao, Xuanmao; Wagner, Erwin; Lisanti, Michael P.; Pestell, Richard G.
Zygotic embryo and shoot tip explants of Phoenix canariensis were cultured on MS (1962) basal medium supplemented with 100 µM Picloram and 9.5 µM kinetin or 10.8 µM or 45.25 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 9.8 µM N6-(2-isopentenyl) adenine (2iP). These explants after 12 weeks in darkness at 28 °C, produced embryogenic callus with very compact, pale yellow, nodular structures.
Le Thi Lan Huong; Michela Baiocco; Bao Phan Huy; Bruno Mezzetti; Rodolfo Santilocchi; Pasquale Rosati
Nanog has an important role in pluripotency induction in bovines and snow leopards. To examine whether it was required for wild felids globally, we examined the induction of pluripotency in felids from Asia (Bengal tiger, Panthera tigris), Africa (serval, Leptailurus serval), and the Americas (jaguar, Panthera onca). Dermal fibroblasts were transduced with genes encoding the human transcription factors OCT4, SOX2, KLF4, and cMYC with or without NANOG. Both four- and five-factor induction resulted in colony formation at day 3 in all three species tested; however, we were not able to maintain colonies that were generated without NANOG beyond passage (P) 7. Five-factor induced pluripotent stem cell (iPSC) colonies from wild cats were expanded in vitro on feeder layers and were positive for alkaline phosphatase and protein expression of OCT-4, NANOG, and stage-specific embryonic antigen-4 at P4 and P14. Reverse-transcription polymerase chain reaction confirmed that all five human transgenes were transcribed at P4; however, OCT4, SOX2, and NANOG transgenes were silenced by P14. Endogenous OCT4 and NANOG transcripts were detected at P4 and P14 in all cell lines confirming successful reprogramming. At P14, the iPSCs from all three species remained euploid and differentiated in vivo and in vitro into derivatives of the three germ layers. This study describes an effective method for inducing pluripotency in three endangered wild cats from across the globe and confirms Nanog as an essential factor in the reprogramming event. Efficient production of iPSC from endangered felids creates a unique opportunity for species preservation through gamete production, nuclear transfer, embryo complementation, and future novel technologies. PMID:23514873
Verma, Rajneesh; Liu, Jun; Holland, Michael Kenneth; Temple-Smith, Peter; Williamson, Mark; Verma, Paul John
Abstract Nanog has an important role in pluripotency induction in bovines and snow leopards. To examine whether it was required for wild felids globally, we examined the induction of pluripotency in felids from Asia (Bengal tiger, Panthera tigris), Africa (serval, Leptailurus serval), and the Americas (jaguar, Panthera onca). Dermal fibroblasts were transduced with genes encoding the human transcription factors OCT4, SOX2, KLF4, and cMYC with or without NANOG. Both four- and five-factor induction resulted in colony formation at day 3 in all three species tested; however, we were not able to maintain colonies that were generated without NANOG beyond passage (P) 7. Five-factor induced pluripotent stem cell (iPSC) colonies from wild cats were expanded in vitro on feeder layers and were positive for alkaline phosphatase and protein expression of OCT-4, NANOG, and stage-specific embryonic antigen-4 at P4 and P14. Reverse-transcription polymerase chain reaction confirmed that all five human transgenes were transcribed at P4; however, OCT4, SOX2, and NANOG transgenes were silenced by P14. Endogenous OCT4 and NANOG transcripts were detected at P4 and P14 in all cell lines confirming successful reprogramming. At P14, the iPSCs from all three species remained euploid and differentiated in vivo and in vitro into derivatives of the three germ layers. This study describes an effective method for inducing pluripotency in three endangered wild cats from across the globe and confirms Nanog as an essential factor in the reprogramming event. Efficient production of iPSC from endangered felids creates a unique opportunity for species preservation through gamete production, nuclear transfer, embryo complementation, and future novel technologies.
Verma, Rajneesh; Liu, Jun; Holland, Michael Kenneth; Temple-Smith, Peter; Williamson, Mark
The unlimited proliferation capacity of embryonic stem cells (ESCs) combined with their pluripotent differentiation potential in various lineages raised great interest in both the scientific community and the public at large with hope for future prospects of regenerative medicine. However, since ESCs are derived from human embryos, their use is associated with significant ethical issues preventing broad studies and therapeutic applications. To get around this bottleneck, Takahashi and Yamanaka have recently achieved the conversion of adult somatic cells into ES-like cells via the forced expression of four transcription factors: Oct3/4, Sox2, Klf4 and c-Myc. This first demonstration attracted public attention and opened a new field of stem cells research with both cognitive – such as disease modeling - and therapeutic prospects. This pioneer work just received the 2012 Nobel Prize in Physiology or Medicine. Many methods have been reported since 2006, for the generation of induced pluripotent stem (iPS) cells. Most strategies currently under use are based on gene delivery via gamma-retroviral or lentiviral vectors; some experiments have also been successful using plasmids or transposons-based systems and few with adenovirus. However, most experiments involve integration in the host cell genome with an identified risk for insertional mutagenesis and oncogenic transformation. To circumvent such risks which are deemed incompatible with therapeutic prospects, significant progress has been made with transgene-free reprogramming methods based on e.g.: sendaï virus or direct mRNA or protein delivery to achieve conversion of adult cells into iPS. In this review we aim to cover current knowledge relating to both delivery systems and combinations of inducing factors including chemicals which are used to generate human iPS cells. Finally, genetic instability resulting from the reprogramming process is also being considered as a safety bottleneck for future clinical translation and stem cell-therapy prospects based on iPS.
Bayart, Emilie; Cohen-Haguenauer, Odile
Human induced pluripotent stem cells (iPSCs) have become an intriguing approach for neurological disease modeling, because neural lineage-specific cell types that retain the donors' complex genetics can be established in vitro. The statistical power of these iPSC-based models, however, is dependent on accurate diagnoses of the somatic cell donors; unfortunately, many neurodegenerative diseases are commonly misdiagnosed in live human subjects. Postmortem histopathological examination of a donor's brain, combined with premortem clinical criteria, is often the most robust approach to correctly classify an individual as a disease-specific case or unaffected control. In this study, we describe iPSCs generated from a skin biopsy collected postmortem during the rapid autopsy of a 75-year-old male, whole body donor, defined as an unaffected neurological control by both clinical and histopathological criteria. These iPSCs were established in a feeder-free system by lentiviral transduction of the Yamanaka factors, Oct3/4, Sox2, Klf4, and c-Myc. Selected iPSC clones expressed both nuclear and surface antigens recognized as pluripotency markers of human embryonic stem cells (hESCs) and were able to differentiate in vitro into neurons and glia. Statistical analysis also demonstrated that fibroblast proliferation was significantly affected by biopsy site, but not donor age (within an elderly cohort). These results provide evidence that autopsy donor-derived fibroblasts can be successfully reprogrammed into iPSCs, and may provide an advantageous approach for generating iPSC-based neurological disease models. PMID:21839145
Hjelm, Brooke E; Rosenberg, Jon B; Szelinger, Szabolcs; Sue, Lucia I; Beach, Thomas G; Huentelman, Matthew J; Craig, David W
Availability of explants with adequate embryogenic competence is one of the most important limitations for the development\\u000a of regenerable cell suspensions in banana. To increase the number and ease of accessibility to potentially embryogenic explants,\\u000a a novel methodology is described by which young male flower clusters isolated from adult plants are induced to form new flower\\u000a buds and proliferate in
Juan Bernardo Pérez-Hernández; Purificación Rosell-García
Hyophorbe lagenicaulis is a critically endangered palm of Mauritius. Zygotic embryos were isolated from seeds and germinated in vitro on MS salts and vitamins containing activated charcoal. When seedlings were pre-treated in vitro for 2 weeks in liquid medium containing 0.05 mg l-1 paclobutrazol, 80% survived the transfer to soil. Three-week-old seedlings were sectioned longitudinally and partially embedded in medium
V. Sarasan; M. Ramsay; A. Roberts
Embryogenesis in plants is a unique process in the sense that it can be initiated from a wide range of cells other than the zygote. Upon stress, microspores or young pollen grains can be switched from their normal pollen development towards an embryogenic pathway, a process called androgenesis. Androgenesis represents an important tool for research in plant genetics and breeding, since androgenic embryos can germinate into completely homozygous, double haploid plants. From a developmental point of view, androgenesis is a rewarding system for understanding the process of embryo formation from single, haploid microspores. Androgenic development can be divided into three main characteristic phases: acquisition of embryogenic potential, initiation of cell divisions, and pattern formation. The aim of this review is to provide an overview of the main cellular and molecular events that characterize these three commitment phases. Molecular approaches such as differential screening and cDNA array have been successfully employed in the characterization of the spatiotemporal changes in gene expression during androgenesis. These results suggest that the activation of key regulators of embryogenesis, such as the BABY BOOM transcription factor, is preceded by the stress-induced reprogramming of cellular metabolism. Reprogramming of cellular metabolism includes the repression of gene expression related to starch biosynthesis and the induction of proteolytic genes (e.g. components of the 26S proteasome, metalloprotease, cysteine, and aspartic proteases) and stress-related proteins (e.g. GST, HSP, BI-1, ADH). The combination of cell tracking systems with biochemical markers has allowed the key switches in the developmental pathway of microspores to be determined, as well as programmed cell death to be identified as a feature of successful androgenic embryo development. The mechanisms of androgenesis induction and embryo formation are discussed, in relation to other biological systems, in special zygotic and somatic embryogenesis. PMID:15928015
Maraschin, S F; de Priester, W; Spaink, H P; Wang, M
Somatic embryogenesis is well established for the economic relevant ornamental crop Cyclamen and thus could supplement the elaborate propagation via seeds. However, the use of somatic embryogenesis for commercial large\\u000a scale propagation is still limited due to physiological disorders and asynchronous development within emerged embryos. To\\u000a overcome these problems, profound knowledge of the physiological processes in Cyclamen embryogenesis is essential.
Christina Rode; Sébastien Gallien; Dimitri Heintz; Alain Van Dorsselaer; Hans-Peter Braun; Traud Winkelmann
During mammalian embryonic development, the primitive streak initiates the differentiation of pluripotent epiblast cells into germ layers. Pluripotency can be reacquired in committed somatic cells using a combination of a handful of transcription factors, such as OCT3/4, SOX2, KLF4 and c-MYC (hereafter referred to as OSKM), albeit with low efficiency. Here we show that during OSKM-induced reprogramming towards pluripotency in human cells, intermediate cells transiently show gene expression profiles resembling mesendoderm, which is a major component of the primitive streak. Based on these findings, we discover that forkhead box H1 (FOXH1), a transcription factor required for anterior primitive streak specification during early development, significantly enhances the reprogramming efficiency of human fibroblasts by promoting their maturation, including mesenchymal to epithelial transition and the activation of late pluripotency markers. These results demonstrate that during the reprogramming process, human somatic cells go through a transient state that resembles mesendoderm. PMID:24759836
Takahashi, Kazutoshi; Tanabe, Koji; Ohnuki, Mari; Narita, Megumi; Sasaki, Aki; Yamamoto, Masamichi; Nakamura, Michiko; Sutou, Kenta; Osafune, Kenji; Yamanaka, Shinya
Somatic embryogenesis from juvenile explants as an efficient way for oak clonal propagation is drastically limited by the low rate of embryo germination. A comparison of the development of immature somatic and zygotic embryos, and a study of the changes in sugar content and lignin accumulation during somatic versus zygotic embryo development were conducted in view of understanding the effect
Magdalena Palada-Nicolau; Jean-François Hausman
The effect of ZnSO4 concentration on barley (Hordeum vulgare L.) microspore embryogenesis was investigated using cultivars of different androgenetic response. Concentrations from 0 (control)\\u000a to 600 ?M in the stress pre-treatment medium alone or in combination with 30 (control) to 600 ?M in the embryo induction medium\\u000a were assayed in anther culture. Incorporation of Zn2+ in the pre-treatment medium itself did not
Begoña Echavarri; Mercedes Soriano; Luis Cistué; M. Pilar Vallés; Ana M. Castillo
Four inhibitors of eukaryotic topoisomerases were investigated for genotoxic effects in the wing spot test of Drosophila melanogaster. As a somatic mutation and recombination test (SMART) this assay assesses mitotic recombination and mutational events of various kinds. We studied camptothecin as a topoisomerase I inhibitor, as well as ellipticine as an intercalating inhibitor and teniposide and etoposide as two non-intercalating inhibitors of topoisomerase II. Wing spots were induced in flies trans-heterozygous for the recessive wing cell markers multiple wing hairs (mwh) and flare (flr3) as well as in flies heterozygous for mwh and the multiply inverted TM3 balancer chromosome. All four compounds proved significantly genotoxic in this test. The spot induction frequencies formally standardized to the millimolar unit of exposure dose decreased in the order camptothecin > teniposide > ellipticine greater, similar etoposide in the mwh/flr3 inversion-free genotype. In the mwh/TM3 genotype, in which mitotic crossing over is suppressed because of the inversion-heterozygosity, the observed spot frequencies were considerably reduced, but to different extents. In this genotype, spot induction by ellipticine was not statistically significant, and it was determined that >99% of the spots are due to mitotic recombination in mwh/flr3 flies. For the other compounds, spot induction in the inversion-heterozygous genotype was significant. The relative contribution of recombination to total spot induction in the inversion-free genotype was 88% for camptothecin. It was significantly lower for the two epipodophyllotoxins teniposide (71%) and etoposide (59%). Only suggestions can be proffered at present as to how these proportions could be related to the primary damage produced by the respective compounds on the chromosomes. PMID:8671755
Frei, H; Würgler, F E
The development of a complete organism from a single cell involves extraordinarily complex orchestration of biological processes that vary intricately across space and time. Systems biology seeks to describe how all elements of a biological system interact in order to understand, model, and ultimately predict aspects of emergent biological processes. Embryogenesis represents an extraordinary opportunity – and challenge – for the application of systems biology. Systems approaches have already been used successfully to study various aspects of development, from complex intracellular networks to 4D models of organogenesis. Going forward, great advancements and discoveries can be expected from systems approaches applied to embryogenesis and developmental biology.
Edelman, Lucas B.; Chandrasekaran, Sriram; Price, Nathan D.
Improvement of the yield of green plants was obtained by decreasing the concentration of ammonium nitrate in the culture medium\\u000a and using glutamine as a nontoxic nitrogen source. Embryoids developed directly from the microspores on such media, and frequently\\u000a green plants were obtained by germination of the embryoids on the induction medium, whereas formation of calli was greatly\\u000a reduced.\\u000a \\u000a Ficoll
Finn Lok Olsen
The primary objective of the project is to improve plant propagation occurring via somatic embryogenesis. To accomplish this objective, the accumulation and depletion of seed specific storage proteins was studied throughout seed development in alfalfa and...
D. A. Stuart
The objective of this study was to improve induction of embryogenesis in wheat microspore culture in order to obtain a high number of regenerable embryos. The arabinogalactan (AG) Larcoll and the arabinogalactan-protein (AGP) from gum arabic were tested on two spring genotypes to see if they could increase microspore viability and induce embryogenesis in the microspore culture. Adding Larcoll significantly
Jocelyne Letarte; Ecaterina Simion; Mai Miner; Ken J. Kasha
Triploid hybrid Citrus plants were regenerated by somatic embryogenesis in vitro from endosperm derived calli. A sequence of media formulations was used to induce and support proliferation of primary callus from endosperm, to induce embryogenesis from primary callus, and to allow embryo development leading to viable plantlets. Calli were induced from cellular endosperm of Citrus sinensis (sweet orange), C. Xparadisi (grapefruit), and C. grandis (pummelo) excised 12-14 weeks post-anthesis. Induction of embryogenesis from sweet orange and pummelo primary calli required gibberellic acid and double mineral nutrient concentrations. Embryogenesis was not induced from grapefruit calli in these experiments. Only sweet orange embryos developed sufficiently to allow plant regeneration. Triploid axillary buds were minigrafted onto etiolated diploid rootstock seedlings in vitro in order to transfer triploid regenerants to soil and the external environment. Triploidy (2n = 3x = 27) was observed consistently in all phases of regeneration and in recovered plants. These results demonstrate that triploid hybrid plant recovery from Citrus endosperm can overcome barriers to sexual hybridization resulting from apomixis. PMID:24221109
Gmitter, F G; Ling, X B; Deng, X X
A novel method for direct organogenesis in onion (Allium cepa L.) resulting in the formation of multiple shoot structures induced on mature flower buds or ovaries in a two-step culture\\u000a procedure is described. Flowers were cultured on an induction medium containing 2 mg\\/l 2,4-dichlorophenoxyacetic acid (2,4-D)\\u000a and 2 mg\\/l 6-benzylaminopurine (BAP). After 6 days (superior to 3 or 12 days),
Z. Luthar; B. Bohanec
Flow cytometry was used to measure the nuclear DNA content of Eucalyptus globulus Labill. somatic and zygotic embryos and leaves in order to determine if somatic embryogenesis induces DNA content and ploidy changes in this species. Mature zygotic embryos derived from open-pollination orchard families were collected from a location in the centre of Portugal. One group was kept for nuclear
G. Pinto; J. Loureiro; T. Lopes; C. Santos
The fusion of Citrus sinensis cv. Hamlin (sweet orange) protoplasts isolated from an embryogenic suspension culture with Severinia disticha (Philippine box orange) protoplasts isolated from epicotyl-derived callus with organogenic potential, resulted in the regeneration of allotetraploid somatic hybrid plants. Plant regeneration was a function of complementation, combining the capacity for somatic embryogenesis of C. sinensis with the organogenic ability of
J. W. Grosser; F. G. Gmitter; J. L. Chandler
\\u000a David Douglas, when he first described Picea sitchensis wrote “It has the great advantage that it thrives on poor soils and could become a large and useful tree in Great Britain”.\\u000a It has since become the most extensively planted exotic species in Great Britain after its introduction by Douglas in 1831\\u000a from its native range in the west coast fog
Allan John; Pascal Drake; Christopher Selby
The results of embryological observation on embryo development in the thirteen cultivars of jujube indicated that they shared the same experience from proembryo to globular embryo, heart-shaped embryo, torpedo-shaped embryo and cotyledon stage embryo. Embryo began aborting from globular or heart-shaped embryo period in most of cultivars of jujube, but in few of them embryo could keep synchronous developing to cotyledon stage embryo. And the courses of embryo development were not synchronous. PMID:17117552
Hao, Jian Ping; Jin, Zhu Ping; Wang, Yong Kang; Zhang, Bao Hua; Li, Deng Ke
Summary Shoot tip cultures from 2- to 3-d-old seedlings ofSorghum bicolor (L.) Moench cv. IS3620C develop highly embryogenic callus from which plants can be regenerated when transferred to plant\\u000a growth regulator-free medium. Isolated shoot tips were cultured on Murashige and Skoog medium supplemented with 2.5 mg\\/liter\\u000a 2,4-dichloro-phenoxyacetic acid and 0.05 mg\\/liter kinetin. Purple pigmentation characteristic of sorghum cultures on growth\\u000a regulator-free
Shyamala Bhaskaran; Roberta H. Smith
Triploid hybrid Citrus plants were regenerated by somatic embryogenesis in vitro from endosperm derived calli. A sequence of media formulations was used to induce and support proliferation of primary callus from endosperm, to induce embryogenesis from primary callus, and to allow embryo development leading to viable plantlets. Calli were induced from cellular endosperm of Citrus sinensis (sweet orange), C. Xparadisi
F. G. Gmitter; X. B. Ling; X. X. Deng
Production of embryos in culture via either somatic embryogenesis or androgenesis has long been used as a propagation tool and as a model system in the investigation of structural, physiological, and molecular events governing embryo development. Despite the similar external morphology to their zygotic counterparts, cultured embryos often fail to develop properly and convert into viable plants during post-embryonic growth. These deficiencies are the results of structural and physiological deviations ascribed to sub-optimal culture conditions. In an attempt to enhance embryo yield and quality we have conducted a series of investigations into the role of glutathione during embryogenesis. Changes in the glutathione redox state represent a key metabolic switch which triggers embryo growth. The imposition of a reduced environment during the early embryonic phases promotes cellular proliferation and increases the number of immature embryos, possibly by promoting the synthesis of nucleotides in support of energetic processes and mitotic activity. Continuation of embryo development is best achieved if the glutathione pool is experimentally switched towards an oxidized state; a condition which favors histodifferentiation and post-embryonic growth in both angiosperm and gymnosperms species. Among the structural events favored by the imposed oxidized environment is the proper formation of the shoot apical meristem (SAM), which acquires a "zygotic-like" appearance. The apical poles of treated embryos are well organized and display a proper expression and localization of meristem marker genes. These conditions are not met in control embryos which form abnormal SAMs characterized by the presence of intercellular spaces and differentiation of meristematic cells. Such meristems fail to reactivate at germination resulting in embryo abortion. Physiological and molecular studies have further demonstrated that the oxidized glutathione environment induces several responses, including changes in ascorbate metabolism, abscisic acid and ethylene synthesis, as well as alterations in storage product deposition patterns. This review attempts to relate these responses to the improved embryonic performance and proposes improved culture conditions to be applied for those cell lines and species recalcitrant to in vitro embryogenesis. PMID:19963394
Background Microspore embryogenesis represents a unique system of single cell reprogramming in plants wherein a highly specialized cell, the microspore, by specific stress treatment, switches its fate towards an embryogenesis pathway. In Brassica napus, a model species for this phenomenon, incubation of isolated microspores at 32°C is considered to be a pre-requisite for embryogenesis induction. Results We have developed a new in vitro system at lower temperature (18°C) to efficiently induce microspore embryogenesis throughout two different developmental pathways: one involving the formation of suspensor-like structures (52.4%) and another producing multicellular embryos without suspensor (13.1%); additionally, a small proportion of non-responsive microspores followed a gametophytic-like development (34.4%) leading to mature pollen. The suspensor-like pathway followed at 18°C involved the establishment of asymmetric identities from the first microspore division and an early polarity leading to different cell fates, suspensor and embryo development, which were formed by cells with different organizations and endogenous auxin distribution, similar to zygotic embryogenesis. In addition, a new strategy for germination of microspore derived embryos was developed for achieving more than 90% conversion of embryos to plantlets, with a predominance of spontaneous doubled haploids plants. Conclusion The present work reveals a novel mechanism for efficient microspore embryogenesis induction in B. napus using continuous low temperature treatment. Results indicated that low temperature applied for longer periods favours an embryogenesis pathway whose first division originates asymmetric cell identities, early polarity establishment and the formation of suspensor-like structures, mimicking zygotic embryogenesis. This new in vitro system provides a convenient tool to analyze in situ the mechanisms underlying different developmental pathways during the microspore reprogramming, breaking or not the cellular symmetry, the establishment of polarity and the developmental embryo patterning, which further produce mature embryos and plants.
Opinion statement There are many new developments regarding somatization disorder, which is among the most difficult and cumbersome of the psychiatric\\u000a disorders encountered in neurology practice. Diagnostic criteria have been revised to facilitate clinical care and research.\\u000a The differential diagnosis includes neurologic disorders (eg, multiple sclerosis, epilepsy), systemic medical disorders, and other psychiatric disorders (eg, mood and anxiety disorders, conversion disorder,
Donald M. Hilty; James A. Bourgeois; Celia H. Chang; Mark E. Servis
The developmental pathway of somatic embryo- genesis in Norway spruce involves proliferation of proembryogenic masses (PEMs), PEM-to-somatic embryo transition and further development of the somatic embryos. It has previously been shown that extracellular signal molecules, including arabino- galactan proteins, lipo-chitooligosaccharides and chitinases, regulate somatic embryogenesis. The Chia4-Pa1 gene from Norway spruce is described here. The Chia4-Pa1 encodes a typical basic
M. Wiweger; I. Farbos; M. Ingouff; U. Lagercrantz; S. von Arnold
With few exceptions, most cells in adult organisms have lost the expression of stem cell-associated proteins and are instead characterized by tissue-specific gene expression and function. This cell fate specification is dictated spatially and temporally during embryogenesis. It has become increasingly apparent that the elegant and complicated process of cell specification is "undone" in cancer. This may be because cancer cells respond to their microenvironment and mutations by acquiring a more permissive, plastic epigenome, or because cancer cells arise from mutated stem cells. Regardless, these advanced cancer cells must use stem cell-associated proteins to sustain their phenotype. One such protein is Nodal, an embryonic morphogen belonging to the transforming growth factor-? (TGF-?) superfamily. First described in early developmental models, Nodal orchestrates embryogenesis by regulating a myriad of processes, including mesendoderm induction, left-right asymmetry and embryo implantation. Nodal is relatively restricted to embryonic and reproductive cell types and is thus absent from most normal adult tissues. However, recent studies focusing on a variety of malignancies have demonstrated that Nodal expression re-emerges during cancer progression. Moreover, in almost every cancer studied thus far, the acquisition of Nodal expression is associated with increased tumourigenesis, invasion and metastasis. As the list of cancers that express Nodal grows, it is essential that the scientific and medical communities fully understand how this morphogen is regulated in both normal and neoplastic conditions. Herein, we review the literature relating to normal and pathological Nodal signalling. In particular, we emphasize the role that this secreted protein plays during morphogenic events and how it signals to support stem cell maintenance and tumour progression. PMID:23291354
Quail, Daniela F; Siegers, Gabrielle M; Jewer, Michael; Postovit, Lynne-Marie
Somatic embryos of Camellia japonica were hydrogel encapsulated using 3% sodium alginate and 0.1 M calcium chloride to produce\\u000a synthetic seeds. Both germinability and repetitive embryogenesis capacity of the encapsulated embryos were investigated. The\\u000a frequency of in vitro germination into plants of artificial seeds was affected by various nutrient additives included in the\\u000a encapsulating matrix. The addition of Ca-free Murashige
Laura V. Janeiro; Antonio Ballester; Ana M. Vieitez
Embryogenic induction in cultures of isolated microspores is a stress-dependent process, which can be triggered by heat shock, sucrose or nitrogen starvation or by anti-microtubular drugs. As they are known to mimic biotic stress, oligosaccharides were tested as an alternative source of compounds to induce microspore embryogenesis in Brassica oleracea var. italica. Among the eight oligosaccharide series that were investigated
C. Lemonnier-Le Penhuizic; C. Chatelet; B. Kloareg; P. Potin
Isolated microspores of Brassica napus are developmentally programmed to form gametes; however, microspores can be reprogrammed through stress treatments to undergo appropriate divisions and form embryos. We are interested in the identification and isolation of factors and genes associated with the induction and establishment of embryogenesis in isolated microspores. Standard and normalized cDNA libraries, as well as subtractive cDNA libraries, were constructed from freshly isolated microspores (0 h) and microspores cultured for 3, 5, or 7 d under embryogenesis-inducing conditions. Library comparison tools were used to identify shifts in metabolism across this time course. Detailed expressed sequence tag analyses of 3 and 5 d cultures indicate that most sequences are related to pollen-specific genes. However, semiquantitative and real-time reverse transcription-polymerase chain reaction analyses at the initial stages of embryo induction also reveal expression of embryogenesis-related genes such as BABYBOOM1, LEAFY COTYLEDON1 (LEC1), and LEC2 as early as 2 to 3 d of microspore culture. Sequencing results suggest that embryogenesis is clearly established in a subset of the microspores by 7 d of culture and that this time point is optimal for isolation of embryo-specific expressed sequence tags such as ABSCISIC ACID INSENSITIVE3, ATS1, LEC1, LEC2, and FUSCA3. Following extensive polymerase chain reaction-based expression profiling, 16 genes were identified as unequivocal molecular markers for microspore embryogenesis in B. napus. These molecular marker genes also show expression during zygotic embryogenesis, underscoring the common developmental pathways that function in zygotic and gametic embryogenesis. The quantitative expression values of several of these molecular marker genes are shown to be predictive of embryogenic potential in B. napus cultivars (e.g. 'Topas' DH4079, 'Allons,' 'Westar,' 'Garrison'). PMID:17384168
Malik, Meghna R; Wang, Feng; Dirpaul, Joan M; Zhou, Ning; Polowick, Patricia L; Ferrie, Alison M R; Krochko, Joan E
This study provides a physiological analysis of somatic embryogenesis in four elite cultivars of date palms: Ahmar, Amsekhsi, Tijib, and Amaside, from the initial callogenesis to establishment and proliferation of embryogenic suspension cultures. Somatic embryos development and in vitro plants rooting were also studied. For each step, auxins and cytokinins concentrations were optimised. The primary callogenesis from leaf explants of seedlings appeared highly dependent on genotype. Ahmar (80%) and Amsekhsi (76%) appeared highly callogenic, whereas Tijib (10%) and Amaside (2%) produced low amounts of calluses. 2,4-Dichlorophenoxyacetic acid appeared favorable to the induction of primary callogenesis and its effect was enhanced by the addition of benzyl adenine or adenine sulfate. Secondary friable calli obtained from chopped granular calli were used to initiate embryogenic cell suspensions in media supplied with 2,4-dichlorophenoxyacetic acid. Suspension cultures showed a growth rate of fourfold after four subcultures in presence of 2,4-dichlorophenoxyacetic acid 2?mg/L. Our results showed that a seven-day transitory treatment with benzyl adenine 0,5?mg/L was necessary to optimize embryos development. Naphthalene acetic acid induced the development of primary orthogravitropic roots during embryos germination. The comparison with cytofluorometry of nuclear DNA amounts showed no significant difference in ploidy level between regenerated plants and seedlings.
Sane, Djibril; Aberlenc-Bertossi, Frederique; Diatta, Leopold Ibrahima Djitiningo; Gueye, Badara; Daher, Abdourahman; Sagna, Maurice; Duval, Yves; Borgel, Alain
Chromatin diminution is the programmed elimination of specific DNA sequences during development. It occurs in diverse species, but the function(s) of diminution and the specificity of sequence loss remain largely unknown. Diminution in the nematode Ascaris suum occurs during early embryonic cleavages and leads to the loss of germline genome sequences and the formation of a distinct genome in somatic cells. We found that ?43 Mb (?13%) of genome sequence is eliminated in A. suum somatic cells, including ?12.7 Mb of unique sequence. The eliminated sequences and location of the DNA breaks are the same in all somatic lineages from a single individual and between different individuals. At least 685 genes are eliminated. These genes are preferentially expressed in the germline and during early embryogenesis. We propose that diminution is a mechanism of germline gene regulation that specifically removes a large number of genes involved in gametogenesis and early embryogenesis. PMID:23123092
Wang, Jianbin; Mitreva, Makedonka; Berriman, Matthew; Thorne, Alicia; Magrini, Vincent; Koutsovoulos, Georgios; Kumar, Sujai; Blaxter, Mark L; Davis, Richard E
Somatic embryogenesis (SE) represents a useful experimental system for studying the regulatory mechanisms of embryo development. In this study, the effect of carbohydrates and osmoticum on storage reserve accumulation and germination of Norway spruce [Picea abies (L.) Karst] somatic embryos were investigated. Using time lapse photography, we monitored development from proliferation of proembryogenic masses (PEMs) to maturation of somatic embryos in two P. abies cell lines cultured on two maturation treatments. A combination of sugar assays, metabolic and proteomic analyses were used to quantify storage reserves in the mature somatic embryos. The maturation treatment containing a nonpermeating osmoticum, polyethylene glycol (PEG, 7.5%) and maltose (3%) as the carbohydrate gave significantly high maturation and low germination frequencies of somatic embryos compared to the treatment with only 3% sucrose. Somatic embryos treated with 3% sucrose contained high levels of sucrose, raffinose and late embryogenesis abundant (LEA) proteins. These compounds are known to be involved in the acquisition of desiccation tolerance during seed development and maturation. In addition the sucrose treatment significantly increased the content of starch in the somatic embryos while the maltose and PEG treatment resulted in somatic embryos with a high content of storage proteins. The high levels of sucrose, raffinose and LEA proteins in the embryos treated with 3% sucrose suggest that sucrose may improve the germination of somatic embryos by promoting the acquisition of desiccation tolerance. PMID:23421376
Businge, Edward; Bygdell, Joakim; Wingsle, Gunnar; Moritz, Thomas; Egertsdotter, Ulrika
Programmed cell death (PCD) in multicellular organisms is a vital process in growth, development, and stress responses that contributes to the formation of tissues and organs. Although numerous studies have defined the molecular participants in apoptotic and PCD cascades, successful identification of early master regulators that target specific cells to live or die is limited. Using Zea mays somatic embryogenesis as a model system, we report that the expressions of two plant hemoglobin (Hb) genes (ZmHb1 and ZmHb2) regulate the cell survival/death decision that influences somatic embryogenesis through their cell-specific localization patterns. Suppression of either of the two ZmHbs is sufficient to induce PCD through a pathway initiated by elevated NO and Zn2+ levels and mediated by production of reactive oxygen species. The effect of the death program on the fate of the developing embryos is dependent on the localization patterns of the two ZmHbs. During somatic embryogenesis, ZmHb2 transcripts are restricted to a few cells anchoring the embryos to the subtending embryogenic tissue, whereas ZmHb1 transcripts extend to several embryonic domains. Suppression of ZmHb2 induces PCD in the anchoring cells, allowing the embryos to develop further, whereas suppression of ZmHb1 results in massive PCD, leading to abortion. We conclude that regulation of the expression of these ZmHbs has the capability to determine the developmental fate of the embryogenic tissue during somatic embryogenesis through their effect on PCD. This unique regulation might have implications for development and differentiation in other species.
Huang, Shuanglong; Hill, Robert D.; Wally, Owen S.D.; Dionisio, Giuseppe; Ayele, Belay T.; Jami, Sravan Kumar; Stasolla, Claudio
Background Although If, encoded by the hyperpolarization-activated cyclic-nucleotide-modulated (HCN) channel gene family, is known to be functionally important in pacing, its mechanistic action is largely inferential and indeed somewhat controversial. To dissect in detail the role of If, we investigated the functional consequences of overexpressing in adult guinea pig left ventricular cardiomyocytes (LVCMs) various HCN1 constructs that have been engineered to exhibit different gating properties. Methods and Results We created the recombinant adenoviruses Ad-CMV-GFP-IRES (CGI), Ad-CGI-HCN1, Ad-CGI-HCN1-???, and Ad-CGI-HCN1-Ins, which mediate ectopic expression of GFP alone, WT, EVY235-7???, and Ins HCN1 channels, respectively; EVY235-7??? and Ins encode channels in which the S3—S4 linkers have been shortened and lengthened to favor and inhibit opening, respectively. Ad-CGI-HCN1, Ad-CGI-HCN1-???, and Ad-CGI-HCN1-Ins, but not control Ad-CGI, transduction of LVCMs led to robust expression of If with comparable densities when fully open (?-22 pA/pF at -140 mV; P>0.05) but distinctive activation profiles (V1/2=-70.8±0.6, -60.4±0.7, and -87.7±0.7 mV; P<0.01, respectively). Whereas control (nontransduced or Ad-CGI—transduced) LVCMs were electrically quiescent, automaticity (206±16 bpm) was observed exclusively in 61% of Ad-HCN1-???—transduced cells that displayed depolarized maximum diastolic potential (-60.6±0.5 versus -70.6±0.6 mV of resting membrane potential of control cells; P<0.01) and gradual phase 4 depolarization (306±32 mV/s) that were typical of genuine nodal cells. Furthermore, spontaneously firing Ad-HCN1-???—transduced LVCMs responded positively to adrenergic stimulation (P<0.05) but exhibited neither overdrive excitation nor suppression. In contrast, the remaining 39% of Ad-HCN1-???—transduced cells exhibited no spontaneous action potentials; however, a single ventricular action potential associated with a depolarized resting membrane potential and a unique, incomplete “phase 4—like” depolarization that did not lead to subsequent firing could be elicited on simulation. Such an intermediate phenotype, similarly observed in 100% of Ad-CGI-HCN— and Ad-CGI-HCN1-Ins—transduced LVCMs, could be readily reversed by ZD7288, hinting at a direct role of If. Correlation analysis revealed the specific biophysical parameters required for If to function as an active membrane potential oscillator. Conclusions Our results not only contribute to a better understanding of cardiac pacing but also may advance current efforts that focus primarily on automaticity induction to the next level by enabling bioengineering of central and peripheral cells that make up the native sinoatrial node.
Xue, Tian; Siu, Chung-Wah; Lieu, Deborah K.; Lau, Chu-Pak; Tse, Hung-Fat; Li, Ronald A.
(1) During embryogenesis of the tick Dermacentor andersoni, amino acid concentrations could be separated into 3 groups. In (a) there was slight increase on day 4 of embryogenesis followed by a sharp drop on days 8 and 12 and increase in newly-hatched larv...
F. N. Boctor M. Y. Kamel
This volume traces the major developments of somatic cell genetics via 47 critical papers on somatic cell hybridization, gene transfer, and mutant mammalian cells. Recognized authorities emphasize the importance of applying the combined approach of cell genetics and recombinant DNA to questions of developments, regulations, and growth of both normal and tumor cells.
Free speech of subjects with somatization and paranoia was analyzed to identify and compare self-concept dimensions reflected in their lexical choices. The somatization disorder group conveyed a sense of negativism, distress, and preoccupation with an uncertain self-identity. The paranoid patients portrayed an artificially positive, grandiose…
Oxman, Thomas E.; And Others
Psychogenesis, considered as a linear sequential process by which psychological influences lead to somatic disturbances, is only a link in a larger bio-psycho-social interactional field. Therefore, in practice, a multilateral approach of the whole person, in his psychological, social and somatic aspects, in health and disease, in his habitual and his therapeutical contacts, should be stressed. It seems unlikely that
R. A. Pierloot
Somatic coliphages are amid several groups of bacteriophages that have been suggested as indicators in water quality assessment. One of the limitations frequently endorsed to somatic coliphages as indicators is that they can replicate in the water environment. This review intends to evaluate the significance of this potential replication. In view of: the threshold densities of somatic coliphages and host bacteria needed for productive infection to occur, the densities of both host cells supporting somatic coliphages replication and these phages in water environments, and the poor contribution of lysogenic induction to the free somatic coliphage numbers in water, it can be concluded that replication of somatic coliphages in waters is very unlikely. Consequently, the contribution of replication in the environment of somatic coliphages is expected to have a non-noticeable influence on the numbers of somatic coliphages detected in water environments. Thus, the replication in the environment should not be argued as a limitation to the use of somatic coliphages as indicators. PMID:19040701
Three hundred and forty adults (including sports players, recreational exercisers, mediators and sedentary controls) completed three inventories purporting to measure cognitive and somatic aspects of anxiety. These were the Cognitive-Somatic Anxiety Questionnaire (CSAQ) devised by Schwartz, Davidson & Goleman (Psychosomatic Medicine, 40, 321-328, 1978), the Worry-Emotionality Scale (WES, Morris, Davis & Hutchens, Journal of Educational Psychology, 73, 541-555, 1981) and the Lehrer-Woolfolk (1982) Anxiety Symptom Questionnaire (LWASQ). Factor analysis of the CSAQ and WES identified distinct cognitive and somatic anxiety factors in both inventories. Higher somatic than cognitive ratings were recorded on the CSAQ and WES, while the pattern was reversed on the LWASQ. The CSAQ can tentatively be recommended as a useful measure of these two anxiety components. We were unable to confirm an observation made previously in the literature that practice of meditation is associated with reduced cognitive anxiety, or that exercise is linked with lower somatic anxiety. PMID:2405835
Steptoe, A; Kearsley, N
In seed plants, embryogenesis is an important process to produce a new generation. It comprises three steps: establishment of organization as an embryo, accumulation of storage substances in the embryo, and acquisition of desiccation tolerance and seed dormancy. These steps are accurately regulated by many factors, including phytohormones, proteins, transcription factors, and other substances related to embryogenesis. The embryogenesis mechanism
Mikihisa Umehara; Miho Ikeda; Hiroshi Kamada
A global DNA methylation and proteomics approach was used to investigate somatic embryo maturation in hybrid larch. Each developmental step during somatic embryogenesis was associated with a distinct and significantly different global DNA methylation level: from 45.8% mC for undifferentiated somatic embryos (1-week proliferation) to 61.5% mC for immature somatic embryos (1-week maturation), while maturation was associated with a decrease in DNA methylation to 53.4% for mature cotyledonary somatic embryos (8-weeks maturation). The presence of 5-azacytidine (hypo-methylating agent) or hydroxyurea (hyper-methylating agent) in the maturation medium altered the global DNA methylation status of the embryogenic cultures, and significantly reduced both their relative growth rate and embryogenic potential, suggesting an important role for DNA methylation in embryogenesis. Maturation was also assessed by examining changes in the total protein profile. Storage proteins, identified as legumin- and vicilin-like, appeared at the precotyledonary stage. In the proteomic study, total soluble proteins were extracted from embryos after 1 and 8 weeks of maturation, and separated by two-dimensional gel electrophoresis. There were 147 spots which showed significant differences between the stages of maturation; they were found to be involved mainly in primary metabolism and the stabilization of the resulting metabolites. This indicated that the somatic embryo was still metabolically active at 8 weeks of maturation. This is the first report of analyses of global DNA methylation (including the effects of hyper- and hypo-treatments) and proteome during somatic embryogenesis in hybrid larch, and thus provides novel insights into maturation of conifer somatic embryos. PMID:23789891
Teyssier, Caroline; Maury, Stéphane; Beaufour, Martine; Grondin, Cécile; Delaunay, Alain; Le Metté, Claire; Ader, Kevin; Cadene, Martine; Label, Philippe; Lelu-Walter, Marie-Anne
In the present investigation, nutritional requirements for induction of a high frequency of well formed somatic embryos (SEs)\\u000a from zygotic embryos (ZEs) of sunflower were assessed. Variables like genotype, embryo size (0.5–10 mm), sucrose concentration\\u000a (30–240 g l?1), carbohydrate source (sucrose, glucose, maltose), agar strength (0.2–1.0%), basal media (MS, Gamborg, Nitsch, White), photoperiod\\u000a (light\\/dark) and temperature (20–36C) were tested. All
M. Sujatha; A. J. Prabakaran
Feijoa (Acca sellowiana, Myrtaceae), a native fruit species from southern Brazil and northern Uruguay, is considered to constitute a reference system\\u000a for somatic embryogenesis in woody dicots. This in vitro regenerative pathway is an efficient micropropagation method, and\\u000a a suitable model system for studies in plant developmental physiology. This study attempts to detect and identify proteins\\u000a that are expressed during
Gabriela Claudia Cangahuala-Inocente; Andrea Villarino; Daniela Seixas; Eliane Dumas-Gaudot; Hernán Terenzi; Miguel Pedro Guerra
The Drosophila mef2 gene encodes a MADS domain transcription factor required for the differentiation of cardiac, somatic, and visceral muscles\\u000a during embryogenesis and the patterning of adult indirect flight muscles assembled during metamorphosis. A prerequisite for\\u000a D-MEF2 function in myogenesis is its precise expression in multiple cell types during development. Novel enhancers for D-mef2 transcription in cardiac and adult muscle
Kathleen Gajewski; Yongsok Kim; Cheol Yong Choi; R. A. Schulz
Differentiating calli derived from rice (Oryza sativa L.) microspores were examined histologically. Shoot and root meristems were observed to be arising by both organogenesis as well as embryogenesis. Embryoid attachment to callus (as well as other embryoids) was at the scutellum adjacent to the mesocotyl and radicle. These observations could be interpreted as an indication of the totipotent plasticity of
A. D. Genovesi; C. W. Magill
Papaya production is seriously limited by Papaya ringspot virus (PRSV) worldwide and Papaya leaf-distortion mosaic virus (PLDMV) in Eastern Asia. An efficient transformation method for developing papaya lines with transgenic resistance to these viruses and commercially desirable traits, such as hermaphroditism, is crucial to shorten the breeding program for this fruit crop. In this investigation, an untranslatable chimeric construct pYP08 containing truncated PRSV coat protein (CP) and PLDMV CP genes coupled with the 3' untranslational region of PLDMV, was generated. Root segments from different portions of adventitious roots of in vitro multiple shoots of hermaphroditic plants of papaya cultivars 'Tainung No. 2', 'Sunrise', and 'Thailand' were cultured on induction medium for regeneration into somatic embryos. The highest frequency of somatic embryogenesis was from the root-tip segments of adventitious roots developed 2-4 weeks after rooting in perlite medium. After proliferation, embryogenic tissues derived from somatic embryos were wounded in liquid-phase by carborundum and transformed by Agrobacterium carrying pYP08. Similarly, another construct pBG-PLDMVstop containing untranslatable CP gene of PLDMV was also transferred to 'Sunrise' and 'Thailand', the parental cultivars of 'Tainung No. 2'. Among 107 transgenic lines regenerated from 349 root-tip segments, nine lines of Tainung No. 2 carrying YP08 were highly resistant to PRSV and PLDMV, and 9 lines (8 'Sunrise' and 1 'Thailand') carrying PLDMV CP highly resistant to PLDMV, by a mechanism of post-transcriptional gene silencing. The hermaphroditic characteristics of the transgenic lines were confirmed by PCR with sex-linked primers and phenotypes of flower and fruit. Our approach has generated transgenic resistance to both PRSV and PLDMV with commercially desirable characters and can significantly shorten the time-consuming breeding programs for the generation of elite cultivars of papaya hybrids. PMID:19943109
Kung, Yi-Jung; Yu, Tsong-Ann; Huang, Chiung-Huei; Wang, Hui-Chin; Wang, Shin-Lan; Yeh, Shyi-Dong
Somatic mutations of the KIT gene have been reported in mast cell diseases and gastrointestinal stromal tumours. Recently, they have also been found in mediastinal and testicular germ cell tumours (TGCTs), particularly in cases with bilateral disease. We screened the KIT coding sequence (except exon 1) for germline mutations in 240 pedigrees with two or more cases of TGCT. No germline mutations were found. Exons 10, 11 and 17 of KIT were examined for somatic mutations in 123 TGCT from 93 multiple-case testicular cancer families. Five somatic mutations were identified; four were missense amino-acid substitutions in exon 17 and one was a 12 bp in-frame deletion in exon 11. Two of seven TGCT from cases with bilateral disease carried KIT mutations compared with three out of 116 unilateral cases (P=0.026). The results indicate that somatic KIT mutations are implicated in the development of a minority of familial as well as sporadic TGCT. They also lend support to the hypothesis that KIT mutations primarily take place during embryogenesis such that primordial germ cells with KIT mutations are distributed to both testes. PMID:15150569
Rapley, E A; Hockley, S; Warren, W; Johnson, L; Huddart, R; Crockford, G; Forman, D; Leahy, M G; Oliver, D T; Tucker, K; Friedlander, M; Phillips, K-A; Hogg, D; Jewett, M A S; Lohynska, R; Daugaard, G; Richard, S; Heidenreich, A; Geczi, L; Bodrogi, I; Olah, E; Ormiston, W J; Daly, P A; Looijenga, L H J; Guilford, P; Aass, N; Fosså, S D; Heimdal, K; Tjulandin, S A; Liubchenko, L; Stoll, H; Weber, W; Einhorn, L; Weber, B L; McMaster, M; Greene, M H; Bishop, D T; Easton, D; Stratton, M R
Transitions between developmental programs during plant embryogenesis are usually characterized by differential gene expression. Soybean (Glycine max (L.) Merr.) somatic embryos from embryogenic suspension cultures germinate poorly on a germination medium even after being grown on medium containing abscisic acid until developmental changes are induced by desiccation. The objectives of this study were to investigate: (i) the relationship between water
Wennuan Liu; David F. Hildebrand; Patricia J. Moore; Glenn B. Collins
Progress on industrial-scale propagation of conifers by somatic embryogenesis has been hampered by the differences in developmental capabilities between cell lines, which are limiting the capture of genetic gains from breeding programs. In this study, we investigated the metabolic events occurring during somatic embryo development in Norway spruce to establish a better understanding of the fundamental metabolic events required for somatic embryo development. Three embryogenic cell lines of Norway spruce (Picea abies (L.) Karst) with different developmental capabilities were studied during somatic embryo development from proliferation of proembryogenic masses to mature somatic embryos. The three different cell lines displayed normal, aberrant and blocked somatic embryo development. Metabolite profiles from four development stages in each of the cell lines were obtained using combined gas chromatography-mass spectrometry. Multivariate discriminant analyses of the metabolic data revealed significant metabolites (P ??? 0.05) for each development stage and transition. The results suggest that endogenous auxin and sugar signaling affects initial stages of somatic embryo development. Furthermore, the results highlight the importance of a timed stress response and the presence of stimulatory metabolites during late stages of embryo development. PMID:22310018
Businge, Edward; Brackmann, Klaus; Moritz, Thomas; Egertsdotter, Ulrika
Somatic embryogenesis is used for vegetative propagation of conifers. Embryogenic cultures can be established from zygotic embryos; however, the embryogenic potential decreases during germination. In Arabidopsis, LEAFY COTYLEDON (LEC) genes are expressed during the embryonic stage, and must be repressed to allow germination. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) causes de-repression of LEC genes. ABSCISIC ACID3 (ABI3) and its Zea mays ortholog VIVIPAROUS1 (VP1) act together with the LEC genes to promote embryo maturation. In this study, we have asked the question whether TSA treatment in a conifer affects the embryogenic potential and the expression of embryogenesis-related genes. We isolated two conifer LEC1-type HAP3 genes, HAP3A and HAP3B, from Picea abies and Pinus sylvestris. A comparative phylogenetic analysis of plant HAP3 genes suggests that HAP3A and HAP3B are paralogous genes originating from a duplication event in the conifer lineage. The expression of HAP3A is high, in both somatic and zygotic embryos, during early embryo development, but decreases during late embryogeny. In contrast, the expression of VP1 is initially low but increases during late embryogeny. After exposure to TSA, germinating somatic embryos of P. abies maintain the competence to differentiate embryogenic tissue, and simultaneously the germination progression is partially inhibited. Furthermore, when embryogenic cultures of P. abies are exposed to TSA during embryo maturation, the maturation process is arrested and the expression levels of PaHAP3A and PaVP1 are maintained, suggesting a possible link between chromatin structure and expression of embryogenesis-related genes in conifers. PMID:21541665
Uddenberg, Daniel; Valladares, Silvia; Abrahamsson, Malin; Sundström, Jens Fredrik; Sundås-Larsson, Annika; von Arnold, Sara
Rapid and reductive cell divisions during embryogenesis require that intracellular structures adapt to a wide range of cell sizes. The mitotic spindle presents a central example of this flexibility, scaling with the dimensions of the cell to mediate accurate chromosome segregation. To determine whether spindle size regulation is achieved through a developmental program or is intrinsically specified by cell size or shape, we developed a system to encapsulate cytoplasm from Xenopus eggs and embryos inside cell-like compartments of defined sizes. Spindle size was observed to shrink with decreasing compartment size, similar to what occurs during early embryogenesis, and this scaling trend depended on compartment volume rather than shape. Thus, the amount of cytoplasmic material provides a mechanism for regulating the size of intracellular structures. PMID:24233724
Good, Matthew C; Vahey, Michael D; Skandarajah, Arunan; Fletcher, Daniel A; Heald, Rebecca
Human embryogenesis is believed to involve an integrated set of complex yet coordinated development of different organs and tissues mediated by the changes in the spatiotemporal expression of many genes. Here, we report a genome-wide expression analysis during wk 4–9 of human embryogenesis, a critical period when most organs develop. About half of all human genes are expressed, and 18.6% of the expressed genes were significantly regulated during this important period. We further identified >5000 regulated genes, most of which previously were not known to be associated with animal development. Our study fills an important gap in mammalian developmental studies by identifying functional pathways involved in this critical but previously not studied period. Our study also revealed that the genes involved here are distinct from those during early embryogenesis, which include three groups of maternal genes. Furthermore, we discovered that genes in a given developmental process are regulated coordinately. This led us to develop an easily searchable database of this entire collection of gene expression profiles, allowing for the identification new genes important for a particular developmental process/pathway and deducing the potential function of a novel gene. The validity of the predictions from the database was demonstrated with two examples through spatiotemporal analyses of the two novel genes. Such a database should serve as a highly valuable resource for the molecular analysis of human development and pathogenesis.—Yi, H., Xue, L., Guo, M.-X. Ma, J., Zeng, Y., Wang, W., Cai, J.-Y. Hu, H.-M., Shu, H.-B. Shi, Y.-B., Li, W.-X. Gene expression atlas for human embryogenesis.
Yi, Hong; Xue, Lu; Guo, Ming-Xiong; Ma, Jian; Zeng, Yan; Wang, Wei; Cai, Jin-Yang; Hu, Hai-Ming; Shu, Hong-Bing; Shi, Yun-Bo; Li, Wen-Xin
Background In Drosophila melanogaster, a pre-mRNA splicing hierarchy controls sexual identity and ultimately leads to sex-specific Doublesex (DSX) transcription factor isoforms. The male-specific DSXM represses genes involved in female development and activates genes involved in male development. Spatial and temporal control of dsx during embryogenesis is not well documented. Results Here we show that DSXM is specifically expressed in subsets of male somatic gonad cells during embryogenesis. Following testis formation, germ cells remain in contact with DSXM-expressing cells, including hub cells and premeiotic somatic cyst cells that surround germ cells during spermatogenesis in larval and adult testes. Conclusion We show that dsx is transcriptionally regulated in addition to being regulated at the pre-mRNA splicing level by the sex determination hierarchy. The dsx locus is spatially controlled by somatic gonad identity. The continuous expression of DSXM in cells contacting the germline suggests an ongoing short-range influence of the somatic sex determination pathway on germ cell development.
Hempel, Leonie U; Oliver, Brian
Richard Shusterman's "Body Consciousness" aims at formulating a theory of somaesthetics and somatic experience. There has indeed been a growing interest in the role of the body in experience. Shusterman examines the arguments of six important writers who have been influential in this discussion. The emphasis on the body is natural for a…
Wolbachia are required for filarial nematode survival and fertility and contribute to the immune responses associated with human filarial diseases. Here we developed whole-mount immunofluorescence techniques to characterize Wolbachia somatic and germline transmission patterns and tissue distribution in Brugia malayi, a nematode responsible for lymphatic filariasis. In the initial embryonic divisions, Wolbachia segregate asymmetrically such that they occupy only a small subset of cells in the developing embryo, facilitating their concentration in the adult hypodermal chords and female germline. Wolbachia are not found in male reproductive tissues and the absence of Wolbachia from embryonic germline precursors in half of the embryos indicates Wolbachia loss from the male germline may occur in early embryogenesis. Wolbachia rely on fusion of hypodermal cells to populate adult chords. Finally, we detect Wolbachia in the secretory canal lumen suggesting living worms may release bacteria and/or their products into their host. PMID:20689574
Landmann, Frédéric; Foster, Jeremy M; Slatko, Barton; Sullivan, William
Background Smad ubiquitination regulatory factor (Smurf) 1 and 2 are E3 ubiquitin ligases originally identified as inhibitors of transforming growth factor beta signaling and are shown to modulate multiple cellular activities. The roles of Smurfs in vertebrate embryogenesis, however, are not completely understood. Results Here we investigate the function of Smurf2 during early Xenopus development. We show that distinctly from Smurf1, overexpression of Smurf2 in presumptive mesoderm interfered with mesoderm induction and caused axial defects, whereas knockdown of Smurf2 with antisense morpholino oligonucleotides resulted in expansion of the mesoderm. These results imply that Smurf2 may modulate nodal-mediated mesodermal induction. Consistently, ventral expression of Smurf2 induced a partial secondary axis with head structures. In the ectoderm, Smurf2 resembled Smurf1 in controlling neural and epidermal marker expression and influencing head formation. Smurf1, but not Smurf2, additionally affected neural tube closure. Interestingly, both Smurfs could enhance as well as repress neural crest markers, implying that they modulate their targets dynamically during neural plate border specification. Conclusion Our data demonstrate that Smurf1 and Smurf2 have overlapping and distinct functionalities during early frog embryogenesis; collectively, they regulate ectodermal and mesodermal induction and patterning to ensure normal development of Xenopus embryos.
Das, Shaonli; Chang, Chenbei
Here we show that the cytosine-5 methyltransferase DNMT2 controls retrotransposon silencing in Drosophila somatic cells. In Drosophila, significant DNMT2-dependent DNA methylation occurs during early embryogenesis. Suppression of white gene silencing by Mt2 (Dnmt2) null mutations in variegated P[w+] element insertions identified functional targets of DNMT2. The enzyme controls DNA methylation at retrotransposons in early embryos and initiates histone H4K20 trimethylation
Sameer Phalke; Olaf Nickel; Diana Walluscheck; Frank Hortig; Maria Cristina Onorati; Gunter Reuter
Brassica napus cultivar Westar is non-embryogenic under all standard protocols for induction of microspore embryogenesis; however, the rare embryos produced in Westar microspore cultures, induced with added brassinosteroids, were found to develop into heritably stable embryogenic lines after chromosome doubling. One of the Westar-derived doubled haploid (DH) lines, DH-2, produced up to 30% the number of embryos as the highly embryogenic B. napus line, Topas DH4079. Expression analysis of marker genes for embryogenesis in Westar and the derived DH-2 line, using real-time reverse transcription-PCR, revealed that the timely expression of embryogenesis-related genes such as LEAFY COTYLEDON1 (LEC1), LEC2, ABSCISIC ACID INSENSITIVE3, and BABY BOOM1, and an accompanying down-regulation of pollen-related transcripts, were associated with commitment to embryo development in Brassica microspores. Microarray comparisons of 7?d cultures of Westar and Westar DH-2, using a B. napus seed-focused cDNA array (10?642 unigenes), identified highly expressed genes related to protein synthesis, translation, and response to stimulus (Gene Ontology) in the embryogenic DH-2 microspore-derived cell cultures. In contrast, transcripts for pollen-expressed genes were predominant in the recalcitrant Westar microspores. Besides being embryogenic, DH-2 plants showed alterations in morphology and architecture as compared with Westar, for example epinastic leaves, non-abscised petals, pale flower colour, and longer lateral branches. Auxin, cytokinin, and abscisic acid (ABA) profiles in young leaves, mature leaves, and inflorescences of Westar and DH-2 revealed no significant differences that could account for the alterations in embryogenic potential or phenotype. Various mechanisms accounting for the increased capacity for embryogenesis in Westar-derived DH lines are considered.
Malik, Meghna R.; Wang, Feng; Dirpaul, Joan M.; Zhou, Ning; Hammerlindl, Joe; Keller, Wilf; Abrams, Suzanne R.; Ferrie, Alison M. R.; Krochko, Joan E.
Investigation of reproductive development of higher plants in spaceflight represents scientific interest first of all with the necessity to work out the plant space technologies for creation of controlled life-support systems. In such systems mainly the higher plants are considered to be an important component that makes it necessary to obtain the several generations of higher plants with their full ontogenesis. As a rule, seeds obtained in three species of the higher plants in a series of experiments differ from the control by some parameters (Merkis, Laurinavichius, 1983; Musgrave et al., 1998; 2000; Levinskikh et all. 1999; Stankovich et al., 2002). It was shown, that immature embryos generated in microgravity were at a range of developmental stage, while the ground control embryos had all reached the premature stage of development (Kuang et al., 2003). Besides, the distinctions in a degree of nutrient substances accumulation in them were revealed (Kuang et al., 2000). Therefore, the elucidation of the possible reasons for distortion of plant reproduction in microgravity demands the further research. In this study we examined embryogenesis of higher plant Brassica rapa L. with an application of slow horizontal clinostats, that allows to deprive the plants the opportunity to perceive the gravitational stimulus. Some plants were clinorotated from the moment sowing of seeds; in other series the experiment plants were placed on clinostats after formation of flower buds. Temporal fixation of the material was used in these experiments, which allow to obtain material for studying of consecutive stages of embryogenesis. The development of 2-21 day-old embryos was studied. Comparative embryological analysis has shown a similarity in the main of process of embryo differentiation produced under clinorotation and in the stationary control. At the early stages of embryogenesis, the distortion in suspensor formation was observed more frequently. Embryos generated in clinorotation variant had a wider range of developmental stages in comparison with the stationary control. At the stage of embryo maturation the various deviations in embryo differentiation were revealed. These distortions were connected both with cotyledon and radicle development. Possible reasons for deviations in the process of embryogenesis in condition of altered gravity are discussed.
Popova, A.; Ivanenko, G.
In response to an assault by foreign organisms, peripheral B cells can change their antibody affinity and isotype by somatically mutating their genomic DNA. The ability of a cell to modify its DNA is exceptional in light of the potential consequences of genetic alterations to cause human disease and cancer. Thus, as expected, this mechanism of antibody diversity is tightly regulated and coordinated through one protein, activation induced deaminase (AID). AID produces diversity by converting cytosine to uracil within the immunoglobulin loci. The deoxyuracil residue is mutagenic when paired with deoxyguanosine, since it mimics thymidine during DNA replication. Additionally, B cells can manipulate the DNA repair pathways so that deoxyuracils are not faithfully repaired. Therefore, an intricate balance exists which is regulated at multiple stages to promote mutation of immunoglobulin genes, while retaining integrity of the rest of the genome. Here we discuss and summarize the current understanding of how AID functions to cause somatic hypermutation.
Maul, Robert W.; Gearhart, Patricia J.
Four embryogenic clones of Norway spruce have been subcultivated and observed over several years to determine the evolution\\u000a of production of mature embryos and to assess the quality of the embryos produced. A wide range of intraclonal quantitative\\u000a and qualitative variability has been observed within this production. Certain morphologic deviations appeared at the immature\\u000a stage and after maturation, such as
J.-L. Fourré; P. Berger; L. Niquet; P. André
Two simple, rapid and efficient protocols for theregeneration of transformed tetraploid lines ofalfalfa (Medicago falcata L.) have beendeveloped and compared. Leaf explants fromembryogenic lines 47\\/1-150 and 47\\/1-5 were inoculatedwith Agrobacterium tumefaciens containingconstructs carrying the nptII selectable markergene and promoter:gusA gene fusions under thecontrol of the CaMV 35S or Arabidopsis cdc2a,CycB1 and CycA2 promoters. In the firstregeneration system (the MSH system),
C. Y. Shao; E. Russinova; A. Iantcheva; A. Atanassov; A. McCormac; D. F. Chen; M. C. Elliott; A. Slater
Hypocotyl segments and zygotic embryos of coriander formed embryogenic calli at frequencies of up to 75% when cultured on MS medium supplemented with 1 mgl-1 2,4-D. Calli were transferred to MS liquid medium with 1 mgl-1 2,4-D to initiate cell suspension cultures. Embryogenic cells became finely dispersible in the medium as the subculture proceeded. Cultures were transferred to a nitrogen
Suk Weon Kim; Mi Kyung Park; Jang R. Liu
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.
Butenko, R. G.; Dmitriyeva, N. N.; Ongko, V.; Basyrova, L. V.
Several economically important tree species belong to the genus Pinus\\u000a and many of them form the ecological base of forest ecosystems. Pine wood is an important raw material\\u000a for the forest industry and many of the pine species have been involved in conventional tree improvement\\u000a programmes. A lot of effort has been made in the development of vegetative propagation methods,\\u000a especially
Hely Häggman; Jaana Vuosku; Tytti Sarjala; Anne Jokela; Karoliina Niemi
This study on species diversity of ectomycorrhiz al fungi mainly concerns oak and conifer forests of the Wes t- ern Himalaya. These forest communities are part of a wide altitudinal transect largely dominated by trees ha v- ing ectomycorrhizal fungi. This feature warrants attention because the transect includes a range of over 15 °C mean annual temperature and widely different
Anitha Karun; E. A. Siril; E. Radha; V. A. Parthasarathy
Summary \\u000a Baloskion tetraphyllum is a member of the Restionaceae and is an important species for the rehabilitation of disused mine sites and wetland areas, and is also highly prized as\\u000a a cut flower. Its use for restoration of disturbed land is, however, severely limited, due to very poor propagation success\\u000a by conventional methods. A study was conducted to evaluate the
M. Panaia; T. Senaratna; K. W. Dixon; K. Sivasithamparam
Fig is one of the most important fruit trees in Egypt. It used to constitute the major source of income for the inhabitants of the western north coast of Egypt. Since 1993 fig cultivations were threatened by a number of factors including virus, insect and mite infections. An efficient system for regeneration and transformation of the common fig Ficus carica L. cultivar Sultani (fresh consumption) was required to conserve fig cultivation in the area. The effect of different combinations of BA and NAA/2,4-D and kinetin on callus formation from leaf segments were studied. Results showed that the best medium for callus formation was MS supplemented with 2.0 mg/l 2,4-D and 0.2 mg/l kinetin. The best plantlet differentiation was obtained at concentrations of 30 mg/l 2iP and 7 mg/l TDZ with 0.25 mg/l NAA (with a regeneration efficiency of 83 and 79%, respectively). On the other hand, the obtained callus failed to induce organogenesis on media containing a combination of BA and kinetin. The highest shoot formation percentage (89%) was obtained when using 2 mg/l TDZ and 4 mg/l 2iP. The highest percentage of shoots forming roots (95%) was obtained when using MS medium supplemented with 1.0 mg/l IBA. Explants were transformed using Agrobacterium and microprojectile bombardment using the plasmid pISV2678 which harbors the gus-intron and bar genes. Results showed that the highest transformation efficiency using the Agrobacterium (17.5%) was obtained when explants were co-cultivated with the bacteria for 30 min. The highest transformation efficiency recorded using the microprojectile bombardment (12%) was obtained with 2.0 ?g DNA per shot at 1,100 psi and a distance of 6 cm repeated twice. The transgenic nature of regenerated plants was confirmed by PCR analysis, histochemical GUS assay and leaf painting assay. PMID:21912211
Soliman, Hemaid Ibrahim; Gabr, Mahdia; Abdallah, Naglaa A
A sunflower genotype (Helianthus annuus L. cv. Florom-328) able to regenerate plants from in vitro cultures was identified by screening hybrids and inbred lines. Protoplasts of this genotype were isolated from dark grown hypocotyls and were cultured in droplets of agarose-solidified V-KM medium covered by liquid V-KM supplemented with naphthaleneacetic acid (NAA) and benzylaminopurine (BAP). One week later colonies were
Sergei Krasnyanski; László Menczel
Crosses were made between three cytoplasmic male-sterile and five restorer sunflower inbred lines. F1 hybrids, including their parents, were studied for their embryogenetic ability. Sterilized seeds were germinated in culture\\u000a tubes on agar-solidified basal medium. Seven days after germination, epidermal layers from hypocotyls were transferred into\\u000a MS and B5 liquid-midia for 5 and 8 days respectively. Then they were transferred
A. R. Bolandi; M. Branchard; G. Alibert; L. Genzbitel; A. Berville; A. Sarrafi
Shoot tips and leafy bud fragments removed from offshoots of adult date palms (Phoenix dactylifera L.) were cultured on a nutrient medium containing the Murashige and Skoog inorganic salts, 453 µM 2,4-dichlorophenoxyacetic acid, 14.8 µM N6-(2-isopentenyl)adenine and 3 g l-1 activated charcoal to develop nodular callus after 8 months of culture. Callus was cultured in agar-solidified and stationary or shaken
J. Veramendi; L. Navarro
Embryogenic callus derived from offshoot tip of date palm (Phoenix dactylifera L.) was transferred to MS medium containing 0, 25, 50, 75, or 100?M AgNO3 combined with 0 or 0.5?M 2iP. Embryogenic callus weight, number of embryos developed and embryo elongation were significantly influenced by the interaction between silver nitrate (AgNO3) and 2iP. In the absence of 2iP, callus weight
Jameel M Al-Khayri; Abdulaziz M Al-Bahrany
Our laboratory investigates systemic autoimmune disease in the context of mouse models of systemic lupus erythematosus (SLE). SLE is associated with high titers of serum autoantibodies of the IgG class that are predominantly directed against nuclear antigens, with pathological manifestations that are considered by many to be characteristic of an immune-complex mediated disease. In this review, we focus on the known and potential roles of somatic mutagenesis in SLE. We will argue that anti-nuclear antibodies (ANA) arise predominantly from nonautoreactive B cells that are transformed into autoreactive cells by the process of somatic hypermutation (SHM), which is normally associated with affinity maturation during the germinal center reaction. We will also discuss the role of SHM in creating antigenic peptides in the V region of the B cell receptor (BCR) and its potential to open an avenue of unregulated T cell help to autoreactive B cells. Finally, we will end this review with new experimental evidence suggesting that spontaneous somatic mutagenesis of genes that regulate B cell survival and activation is a rate-limiting causative factor in the development of ANA. PMID:23249093
Detanico, Thiago; St Clair, James B; Aviszus, Katja; Kirchenbaum, Greg; Guo, Wenzhong; Wysocki, Lawrence J
In mammals, both parental genomes undergo dramatic epigenetic changes after fertilization to form the diploid somatic genome. Here we show that the paternal genome in the mouse is significantly and actively demethylated within 6-8 hours of fertilization, before the onset of DNA replication, whereas the maternal genome is demethylated after several cleavage divisions. This active demethylation of the paternal genome
Wolfgang Mayer; Alain Niveleau; Jörn Walter; Reinald Fundele; Thomas Haaf
During embryogenesis a single cell gives rise to a functional multicellular organism. In higher plants, as in many other multicellular systems, essential architectural features, such as body axes and major tissue layers are established early in embryogenesis and serve as a positional framework for subsequent pattern elaboration. In Arabidopsis, the apicalbasal axis and the radial pattern of tissues wrapped around it are already recognizable in young embryos of only about a hundred cells in size. This early axial pattern seems to provide a coordinate system for the embryonic initiation of shoot and root. Findings from genetic studies in Arabidopsis are revealing molecular mechanisms underlying the initial establishment of the axial core pattern and its subsequent elaboration into functional shoots and roots. The genetic programs operating in the early embryo organize functional cell patterns rapidly and reproducibly from minimal cell numbers. Understanding their molecular details could therefore greatly expand our ability to generate plant body patterns de novo, with important implications for plant breeding and biotechnology.
Capron, Arnaud; Chatfield, Steven; Provart, Nicholas; Berleth, Thomas
Embryogenesis offers a real laboratory for pattern formation, buckling, and postbuckling induced by growth of soft tissues. Each part of our body is structured in multiple adjacent layers: the skin, the brain, and the interior of organs. Each layer has a complex biological composition presenting different elasticity. Generated during fetal life, these layers will experience growth and remodeling in the early postfertilization stages. Here, we focus on a herringbone pattern occurring in fetal intestinal tissues. Common to many mammalians, this instability is a precursor of the villi, finger-like projections into the lumen. For avians (chicks’ and turkeys’ embryos), it has been shown that, a few days after fertilization, the mucosal epithelium of the duodenum is smooth, and then folds emerge, which present 2 d later a pronounced zigzag instability. Many debates and biological studies are devoted to this specific morphology, which regulates the cell renewal in the intestine. After reviewing experimental results about duodenum morphogenesis, we show that a model based on simplified hypothesis for the growth of the mesenchyme can explain buckling and postbuckling instabilities. Being completely analytical, it is based on biaxial compressive stresses due to differential growth between layers and it predicts quantitatively the morphological changes. The growth anisotropy increasing with time, the competition between folds and zigzags, is proved to occur as a secondary instability. The model is compared with available experimental data on chick’s duodenum and can be applied to other intestinal tissues, the zigzag being a common and spectacular microstructural pattern of intestine embryogenesis.
Ben Amar, Martine; Jia, Fei
As we previously reported for glutathione (GSH), both ascorbic acid (AA) and vitamin E were observed to suppress wild carrot (Daucus carota L.) somatic embryogenesis with little concomitant effect on biomass. Endogenous concentrations of AA were lower during embryo development than during cell proliferation, exhibiting a temporal pattern nearly identical to that of GSH. GSSG (oxidized GSH) reductase was found to be considerably more active in proliferating than in developing cultures, whereas no difference was evident in the case of dehydroascorbate (DHA) reductase. Both GSH and AA concentrations in these cells are governed by 2,4-D. These results show that redox status is a strong determinant of proliferative versus developmental growth and indicate that the mode of action of 2,4-D in this system may be explained at least in part by its influence on endogenous antioxidant levels.
Earnshaw, Brent A.; Johnson, Morris A.
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.
Background Describing the patterns of gene expression during embryonic development has broadened our understanding of the processes and patterns that define morphogenesis. Yet gene expression patterns have not been described throughout vertebrate embryogenesis. This study presents statistical analyses of gene expression during all 40 developmental stages in the teleost Fundulus heteroclitus using four biological replicates per stage. Results Patterns of gene expression for 7,000 genes appear to be important as they recapitulate developmental timing. Among the 45% of genes with significant expression differences between pairs of temporally adjacent stages, significant differences in gene expression vary from as few as five to more than 660. Five adjacent stages have disproportionately more significant changes in gene expression (> 200 genes) relative to other stages: four to eight and eight to sixteen cell stages, onset of circulation, pre and post-hatch, and during complete yolk absorption. The fewest differences among adjacent stages occur during gastrulation. Yet, at stage 16, (pre-mid-gastrulation) the largest number of genes has peak expression. This stage has an over representation of genes in oxidative respiration and protein expression (ribosomes, translational genes and proteases). Unexpectedly, among all ribosomal genes, both strong positive and negative correlations occur. Similar correlated patterns of expression occur among all significant genes. Conclusions These data provide statistical support for the temporal dynamics of developmental gene expression during all stages of vertebrate development.
SLD5 forms a GINS complex with PSF1, PSF2 and PSF3, which is essential for the initiation of DNA replication in lower eukaryotes. Although these components are conserved in mammals, their biological function is unclear. We show here that targeted disruption of SLD5 in mice causes a defect in cell proliferation in the inner cell mass, resulting in embryonic lethality at the peri-implantation stage, indicating that SLD5 is essential for embryogenesis. Moreover, this phenotype of SLD5 mutant mice is quite similar compared with that of PSF1 mutant mice. We have previously reported that haploinsufficiency of PSF1 resulted in failure of acute proliferation of bone marrow hematopoietic stem cells (HSCs) during reconstitution of bone marrow ablated by 5-FU treatment. Since SLD5 was highly expressed in bone marrow, we investigated its involvement in bone marrow reconstitution after bone marrow ablation as observed in PSF1 heterozygous mutant mice. However, heterozygous deletion of the SLD5 gene was found not to significantly affect bone marrow reconstitution. On the other hand, abundant SLD5 expression was observed in human cancer cell lines and heterozygous deletion of the gene attenuated tumor progression in a murine model of spontaneous gastric cancer. These indicated that requirement and dependency of SLD5 for cell proliferation is different in different cell types.
Nagahama, Yumi; Gong, Zhi-Yuan; Asano, Masahide; Oshima, Hiroko; Oshima, Masanobu; Fujio, Yasushi; Takakura, Nobuyuki
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. PMID:24762628
Kuntz, Steven G; Eisen, Michael B
Temperature affects both the timing and outcome of animal development, but the detailed effects of temperature on the progress of early development have been poorly characterized. To determine the impact of temperature on the order and timing of events during Drosophila melanogaster embryogenesis, we used time-lapse imaging to track the progress of embryos from shortly after egg laying through hatching at seven precisely maintained temperatures between 17.5°C and 32.5°C. We employed a combination of automated and manual annotation to determine when 36 milestones occurred in each embryo. D. melanogaster embryogenesis takes 33 hours at 17.5°C, and accelerates with increasing temperature to a low of 16 hours at 27.5°C, above which embryogenesis slows slightly. Remarkably, while the total time of embryogenesis varies over two fold, the relative timing of events from cellularization through hatching is constant across temperatures. To further explore the relationship between temperature and embryogenesis, we expanded our analysis to cover ten additional Drosophila species of varying climatic origins. Six of these species, like D. melanogaster, are of tropical origin, and embryogenesis time at different temperatures was similar for them all. D. mojavensis, a sub-tropical fly, develops slower than the tropical species at lower temperatures, while D. virilis, a temperate fly, exhibits slower development at all temperatures. The alpine sister species D. persimilis and D. pseudoobscura develop as rapidly as tropical flies at cooler temperatures, but exhibit diminished acceleration above 22.5°C and have drastically slowed development by 30°C. Despite ranging from 13 hours for D. erecta at 30°C to 46 hours for D. virilis at 17.5°C, the relative timing of events from cellularization through hatching is constant across all species and temperatures examined here, suggesting the existence of a previously unrecognized timer controlling the progress of embryogenesis that has been tuned by natural selection as each species diverges.
Kuntz, Steven G.; Eisen, Michael B.
We analyzed DNA sequences that regulate the expression of an isocitrate lyase gene from Brassica napus L. during late embryogenesis and during postgerminative growth to determine whether glyoxysomal function is induced by a common mechanism at different developmental stages. beta-Glucuronidase constructs were used both in transient expression assays in B. napus and in transgenic Arabidopsis thaliana to identify the segments of the isocitrate lyase 5' flanking region that influence promoter activity. DNA sequences that play the principal role in activating the promoter during post-germinative growth are located more than 1,200 bp upstream of the gene. Distinct DNA sequences that were sufficient for high-level expression during late embryogenesis but only low-level expression during postgerminative growth were also identified. Other parts of the 5' flanking region increased promoter activity both in developing seed and in seedlings. We conclude that a combination of elements is involved in regulating the isocitrate lyase gene and that distinct DNA sequences play primary roles in activating the gene in embryos and in seedlings. These findings suggest that different signals contribute to the induction of glyoxysomal function during these two developmental stages. We also showed that some of the constructs were expressed differently in transient expression assays and in transgenic plants.
Zhang, J Z; Santes, C M; Engel, M L; Gasser, C S; Harada, J J
Background Morphogenetic events that shape the Drosophila melanogaster embryo are tightly controlled by a genetic program in which specific sets of genes are up-regulated. We used a suppressive subtractive hybridization procedure to identify a group of developmentally regulated genes during early stages of D. melanogaster embryogenesis. We studied the spatiotemporal activity of these genes in five different intervals covering 12 stages of embryogenesis. Results Microarrays were constructed to confirm induction of expression and to determine the temporal profile of isolated subtracted cDNAs during embryo development. We identified a set of 118 genes whose expression levels increased significantly in at least one developmental interval compared with a reference interval. Of these genes, 53% had a phenotype and/or molecular function reported in the literature, whereas 47% were essentially uncharacterized. Clustering analysis revealed demarcated transcript groups with maximum gene activity at distinct developmental intervals. In situ hybridization assays were carried out on 23 uncharacterized genes, 15 of which proved to have spatiotemporally restricted expression patterns. Among these 15 uncharacterized genes, 13 were found to encode putative secreted and transmembrane proteins. For three of them we validated our protein sequence predictions by expressing their cDNAs in Drosophila S2R+ cells and analyzed the subcellular distribution of recombinant proteins. We then focused on the functional characterization of the gene CG6234. Inhibition of CG6234 by RNA interference resulted in morphological defects in embryos, suggesting the involvement of this gene in germ band retraction. Conclusion Our data have yielded a list of developmentally regulated D. melanogaster genes and their expression profiles during embryogenesis and provide new information on the spatiotemporal expression patterns of several uncharacterized genes. In particular, we recovered a substantial number of unknown genes encoding putative secreted and transmembrane proteins, suggesting new components of signaling pathways that might be incorporated within the existing regulatory networks controlling D. melanogaster embryogenesis. These genes are also good candidates for additional targeted functional analyses similar to those we conducted for CG6234. See related minireview by Vichas and Zallen:
Zuniga, Alejandro; Hodar, Christian; Hanna, Patricia; Ibanez, Freddy; Moreno, Pablo; Pulgar, Rodrigo; Pastenes, Luis; Gonzalez, Mauricio; Cambiazo, Veronica
Based on a protocol for microspore culture in apple ( Malus domestica Borkh.), the embryo induction phase has been improved with regard to pretreatment of microspores for initiation of microspore embryogenesis, the concentration of carbon source in the induction medium and the microspore density in the suspension. Furthermore, the effect of the genotype was studied. To determine the efficiency of
The development of fully differentiated plants from individual pollen grains through a series of developmental phases that resemble embryogenesis beginning with the zygote was demonstrated during the mid-1960's. This technology opened the door to the use of haploid plants (sporophytes with the gametic number of chromosomes) for plant breeding and genetic studies, biochemical and metabolic studies, and the selection of mutations. Although pollen embryogenesis has been demonstrated successfully in numerous plant genera, the procedure cannot as yet be used routinely to generate large populations of plants for experiments. Practical results from use of the technology in genetic toxicology research to detect mutations have failed to fully realize the theoretical potential; further developments of the technology could overcome the limitations. Pollen embryogenesis could be used to develop plants from mutant pollen grains to verify that genetic changes are involved. Through either spontaneous or induced chromosome doubling, these plants can be made homozygous and used to analyze genetically the mutants involved. The success of this approach will depend on the mutant frequency relative to the fraction of pollen grains that undergo embryogenesis; these two factors will dictate population size needed for success. Research effort is needed to further develop pollen embryogenesis for use in the detection of genotoxins under both laboratory and in situ conditions.
Background The standard textbook information that annelid musculature consists of oligochaete-like outer circular and inner longitudinal muscle-layers has recently been called into question by observations of a variety of complex muscle systems in numerous polychaete taxa. To clarify the ancestral muscle arrangement in this taxon, we compared myogenetic patterns during embryogenesis of Ophryotrocha diadema with available data on oligochaete and polychaete myogenesis. This work addresses the conflicting views on the ground pattern of annelids, and adds to our knowledge of the evolution of lophotrochozoan taxa. Results Somatic musculature in Ophryotrocha diadema can be classified into the trunk, prostomial/peristomial, and parapodial muscle complexes. The trunk muscles comprise strong bilateral pairs of distinct dorsal and ventral longitudinal strands. The latter are the first to differentiate during myogenesis. They originate within the peristomium and grow posteriorly through the continuous addition of myocytes. Later, the longitudinal muscles also expand anteriorly and form a complex arrangement of prostomial muscles. Four embryonic parapodia differentiate in an anterior-to-posterior progression, significantly contributing to the somatic musculature. Several diagonal and transverse muscles are present dorsally. Some of the latter are situated external to the longitudinal muscles, which implies they are homologous to the circular muscles of oligochaetes. These circular fibers are only weakly developed, and do not appear to form complete muscle circles. Conclusion Comparison of embryonic muscle patterns showed distinct similarities between myogenetic processes in Ophryotrocha diadema and those of oligochaete species, which allows us to relate the diverse adult muscle arrangements of these annelid taxa to each other. These findings provide significant clues for the interpretation of evolutionary changes in annelid musculature.
Bergter, Annette; Brubacher, John L; Paululat, Achim
Carrot cell cultures were used to study the dynamics of calmodulin protein levels, calmodulin methylation, and calmodulin-binding proteins during plant growth and development. Comparisons of proliferating and nonproliferating wild carrot cells show that, while calmodulin protein levels does not vary significantly, substantial variation in post-translational methylation of calmodulin on lysine-115 is observed. Calmodulin methylation is low during the lag and early exponential stages, but increases substantially as exponential growth proceeds and becomes maximal in the postexponential phase. Unmethylated calmodulin quickly reappears within 12 h of reinoculation of cells into fresh media, suggesting that the process is regulated according to the cell growth state. Calmodulin and calmodulin-binding proteins were also analyzed during the formation and germination of domestic carrot embryos in culture. Neither calmodulin methylation nor calmodulin protein levels varied significantly during somatic embryogenesis. However, upon germination of embryos, the level of calmodulin protein doubled. By calmodulin overlay analysis, we have detected a major 54,000 M(r) calmodulin-binding protein that also increased during embryo germination. This protein was purified from carrot embryo extracts by calmodulin-Sepharose chromatography. Overall, the data suggest that calmodulin methylation is regulated depending upon the state of cell growth and that calmodulin and its target proteins are modulated during early plant development. PMID:1637181
Oh, S H; Steiner, H Y; Dougall, D K; Roberts, D M
Third instar larvae of Drosophila melanogaster transdihybrid for mwh and flr were exposed to varying concentrations of cisplatin by feeding on dry media wetted with aqueous solutions of the test compound. Larval feeding continued until pupation, and surviving transdihybrid adults were collected seven days following commencement of feeding. Wings of adults were removed and scored under 400X magnification for the presence of twin spots and single spots comprised of clones of cells possessing malformed wing hairs. Cisplatin was found to induce both twin spots and single spots, and significant linear concentration-response relationships were obtained with respect to the induction of all endpoints. This capacity to induce mitotic exchange in the somatic tissue of Drosophila compares well with the compound's reported ability to induce chromosome breaks in Drosophila germ cells. However, not all compounds possess similar genotoxic profiles in the somatic an germ tissue of Drosophila.
Under specific stress treatments (cold, starvation), in vitro microspores can be induced to deviate from their gametophytic development and switch to embryogenesis, forming haploid embryos and homozygous breeding lines in a short period of time. The inductive stress produces reactive oxygen species (ROS) and nitric oxide (NO), signalling molecules mediating cellular responses, and cell death, modifying the embryogenic microspore response and therefore, the efficiency of the process. This work analysed cell death, caspase 3-like activity, and ROS and NO production (using fluorescence probes and confocal analysis) after inductive stress in barley microspore cultures and embryogenic suspension cultures, as an in vitro system which permitted easy handling for comparison. There was an increase in caspase 3-like activity and cell death after stress treatment in microspore and suspension cultures, while ROS increased in non-induced microspores and suspension cultures. Treatments of the cultures with a caspase 3 inhibitor, DEVD-CHO, significantly reduced the cell death percentages. Stress-treated embryogenic suspension cultures exhibited high NO signals and cell death, while treatment with S-nitrosoglutathione (NO donor) in control suspension cultures resulted in even higher cell death. In contrast, in microspore cultures, NO production was detected after stress, and, in the case of 4-day microspore cultures, in embryogenic microspores accompanying the initiation of cell divisions. Subsequent treatments of stress-treated microspore cultures with ROS and NO scavengers resulted in a decreasing cell death during the early stages, but later they produced a delay in embryo development as well as a decrease in the percentage of embryogenesis in microspores. Results showed that the ROS increase was involved in the stress-induced programmed cell death occurring at early stages in both non-induced microspores and embryogenic suspension cultures; whereas NO played a dual role after stress in the two in vitro systems, one involved in programmed cell death in embryogenic suspension cultures and the other in the initiation of cell division leading to embryogenesis in reprogrammed microspores. PMID:22197894
Rodríguez-Serrano, María; Bárány, Ivett; Prem, Deepak; Coronado, María-José; Risueño, María C; Testillano, Pilar S
The myocyte enhancer factor (MEF) 2 family of transcription factors has been implicated in the regulation of muscle transcription in vertebrates. We have cloned a protein from Drosophila, termed D-MEF2, that shares extensive amino acid homology with the MADS (MCM1, Agamous, Deficiens, and serum-response factor) domains of the vertebrate MEF2 proteins. D-mef2 gene expression is first detected during Drosophila embryogenesis within mesodermal precursor cells prior to specification of the somatic and visceral muscle lineages. Expression of D-mef2 is dependent on the mesodermal determinants twist and snail but independent of the homeobox-containing gene tinman, which is required for visceral muscle and heart development. D-mef2 expression precedes that of the MyoD homologue, nautilus, and, in contrast to nautilus, D-mef2 appears to be expressed in all somatic and visceral muscle cell precursors. Its temporal and spatial expression patterns suggest that D-mef2 may play an important role in commitment of mesoderm to myogenic lineages. Images
Lilly, B; Galewsky, S; Firulli, A B; Schulz, R A; Olson, E N
Somatic treatments for mood disorders represent a class of interventions available either as a stand-alone option, or in combination with psychopharmacology and\\/or psychotherapy. Here, we review the currently available techniques, including those already in clinical use and those still under research. Techniques are grouped into the following categories: (1) seizure therapies, including electroconvulsive therapy and magnetic seizure therapy, (2) noninvasive
Moacyr A Rosa; Sarah H Lisanby
In the genome of a germ-line cell, the genetic information for an immunoglobulin polypeptide chain is contained in multiple gene segments scattered along a chromosome. During the development of bone marrow-derived lymphocytes, these gene segments are assembled by recombination which leads to the formation of a complete gene. In addition, mutations are somatically introduced at a high rate into the
Philosophers of education need a methodology for ensuring that what they teach does not contain cultural evils. What is needed is a criterion by which cultural practices can be evaluated so that educators know what to teach. Somatic knowledge can help in making these distinctions. (Contains 17 references.) (Author)
The transformation of a normal cell into a cancer cell takes place through a sequence of a small number of discrete genetic events, somatic mutations: thus, cancer can be regarded properly as a genetic disease of somatic cells. The analogy between evolution of organisms and evolution of cell populations is compelling: in both cases what drives change is mutation, but it is Darwinian selection that enables clones that have a growth advantage to expand, thus providing a larger target size for the next mutation to hit. The search for molecular lesions in tumors has taken on a new dimension thanks to two powerful technologies: the micro-arrays for quantitative analysis of global gene expresssion (the transcriptome); and 'deep' sequencing for the global analysis of the entire genome (or at least the exome). The former offers the most complete phenotypic characterization of a tumor we could ever hope for--we could call this the ultimate phenotype; the latter can identify all the somatic mutations in an individual tumor--we could call this the somatic genotype. However, there is definitely the risk that while we are 'drowned by data, we remain thirsty for knowledge'. If we want to heed the teachings of Lorenzo Tomatis, I think the message is clear: we ought to take advantage of the new powerful technologies--not by becoming their slaves, but remaining their masters. Identifying somatic mutations in a tumor is important not because it qualifies for 'oncogenomics', but because through a deeper understanding of the nature of that particular tumor it can help us to optimize therapy or to design new therapeutic approaches. PMID:21489208
The transformation of a normal cell into a cancer cell takes place through a sequence of a small number of discrete genetic events, somatic mutations: thus, cancer can be regarded properly as a genetic disease of somatic cells. The analogy between evolution of organisms and evolution of cell populations is compelling: in both cases what drives change is mutation, but it is Darwinian selection that enables clones that have a growth advantage to expand, thus providing a larger target size for the next mutation to hit. The search for molecular lesions in tumors has taken on a new dimension thanks to two powerful technologies: the micro-arrays for quantitative analysis of global gene expresssion (the transcriptome); and ‘deep’ sequencing for the global analysis of the entire genome (or at least the exome). The former offers the most complete phenotypic characterization of a tumor we could ever hope for – we could call this the ultimate phenotype; the latter can identify all the somatic mutations in an individual tumor – we could call this the somatic genotype. However, there is definitely the risk that while we are ‘drowned by data, we remain thirsty for knowledge’. If we want to heed the teachings of Lorenzo Tomatis, I think the message is clear: we ought to take advantage of the new powerful technologies – not by becoming their slaves, but remaining their masters. Identifying somatic mutations in a tumor is important not because it qualifies for ‘oncogenomics’, but because through a deeper understanding of the nature of that particular tumor it can help us to optimize therapy or to design new therapeutic approaches.
A simple and reproducible protocol for induction, growth and development of somatic embryos from hypocotyl explants of Indian\\u000a mustard (Brassica juncea L. Czern & Coss) var. RLM 198 is reported. The HDSE (Hypocotyl-derived somatic embryos) were fleshy globular to torpedo structures\\u000a that were maintained by regular subculturing every three weeks. These embryos developed non-synchronously into the heart shaped-stage\\u000a while some
Anita Kumari; G. S. Cheema; S. K. Munshi
Genetic mutations causing human disease are conventionally thought to be inherited through the germ line from one's parents and present in all somatic (body) cells, except for most cancer mutations, which arise somatically. Increasingly, somatic mutations are being identified in diseases other than cancer, including neurodevelopmental diseases. Somatic mutations can arise during the course of prenatal brain development and cause neurological disease-even when present at low levels of mosaicism, for example-resulting in brain malformations associated with epilepsy and intellectual disability. Novel, highly sensitive technologies will allow more accurate evaluation of somatic mutations in neurodevelopmental disorders and during normal brain development. PMID:23828942
Poduri, Annapurna; Evrony, Gilad D; Cai, Xuyu; Walsh, Christopher A
The chromosomes of one or other of the ``parents'' of a hybrid cell are usually gradually lost over successive generations. X-irradiation, gamma-irradiation or BUDR labelling of one ``parent'' before fusion can be used to predetermine which of the two sets of chromosomes will be preferentially lost.
Abstract All multicellular living beings are created from a single cell. A developmental process, called embryogenesis, takes this first fertilized cell down a complex path of reproduction, migration, and specialization into a complex organism adapted to its environment. In most cases, the first steps of the embryogenesis take place in a protected environment such as in an egg or in utero. Starting from this observation, we propose a new approach to the generation of real robots, strongly inspired by living systems. Our robots are composed of tens of specialized cells, grown from a single cell using a bio-inspired virtual developmental process. Virtual cells, controlled by gene regulatory networks, divide, migrate, and specialize to produce the robot's body plan (morphology), and then the robot is manually built from this plan. Because the robot is as easy to assemble as Lego, the building process could be easily automated. PMID:24730763
Cussat-Blanc, Sylvain; Pollack, Jordan
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.
The origin of somatic embryos obtained from meristematic apexes of the Musa (AAA) clone "Gran enano" was analyzed through histological and morphological studies during the various development phases of the process. The research point out that somatic embryos developed directly from perivascular parenchyma cells of the leaves. Histological sections of globular embryos showed a radial disposition to cell and the existence of an epidermal layer that surrounds the embryo completely. When citocinine (Z or BA) was added, some embryos remained in globular stage with mild signs of enlargement but with no later development of invagination. Other's embryos reached the invagination stage; and some reached the enlargement stage with active photosynthetic tissues. However there were no to generation of complete plant regardless of additional treatment, such as "osmotic shock" or the additions of GA3--At present do not have an explanations for this results. Therefore, additional experiment should be in early, intermediate and later stages of somatic embryogenesis, in order to understand the mechanisms underlying the lack of development of plants from somatics embryos. PMID:11220222
Vidal, M C; Vargas, T E; de García, E
?-Tocopherol is a lipid-soluble antioxidant that is specifically required for reproduction and embryogenesis. However, since its discovery, ?-tocopherol's specific biologic functions, other than as an antioxidant, and the mechanism(s) mediating its requirement for embryogenesis remain unknown. As an antioxidant, ?-tocopherol protects polyunsaturated fatty acids (PUFAs) from lipid peroxidation. ?-Tocopherol is probably required during embryonic development to protect PUFAs that are crucial to development, specifically arachidonic (ARA) and docosahexaenoic (DHA) acids. Additionally, ARA and DHA are metabolized to bioactive lipid mediators via lipoxygenase enzymes, and ?-tocopherol may directly protect, or it may mediate the production and/or actions of, these lipid mediators. In this review, we discuss how ?-tocopherol (1) prevents the nonspecific, radical-mediated peroxidation of PUFAs, (2) functions within a greater antioxidant network to modulate the production and/or function of lipid mediators derived from 12- and 12/15-lipoxygenases, and (3) modulates 5-lipoxygenase activity. The application and implication of such interactions are discussed in the context of ?-tocopherol requirements during embryogenesis. PMID:23920314
Lebold, Katie M; Traber, Maret G
As a product of asexual reproduction in plants, the somatic embryo (SE) differentiates into a new plantlet via a zygotic embryogenesis-like process. Here, we present the phenotypic and cellular differences between SEs and zygotic embryos (ZEs) revealed by histological section scanning using three parallel development stages of the two types of embryos of cotton (Gossypium hirsutum cv. YZ1), including globular, torpedo and cotyledonary-stages. To identify the molecular characteristics of SE development in cotton, the digital gene expression system was used to profile the genes active during SE and ZE development. A total of 4242 differentially expressed genes (DEGs) were identified in at least one developmental stage. Expression pattern and functional classification analysis based on these DEGs reveals that SE development exhibits a transcriptional activation of stress responses. RT-PCR analysis further confirmed enhanced expression levels of stress-related genes in SEs than in ZEs. Experimental stress treatment, induced by NaCl and ABA, accelerated SE development and increased the transcription of genes related to stress response, in parallel with decelerated proliferation of embryogenic calluses under stress treatment. Our data reveal that SE development involves the activation of stress responses, which we suggest may regulate the balance between cell proliferation and differentiation. These results provide new insight into the molecular mechanisms of SE development and suggest strategies that can be used for regulating the developmental processes of somatic embryogenesis. PMID:24112122
Jin, Fangyan; Hu, Lisong; Yuan, Daojun; Xu, Jiao; Gao, Wenhui; He, Liangrong; Yang, Xiyan; Zhang, Xianlong
Stress-induced plant cell reprogramming involves changes in global genome organization, being the epigenetic modifications key factors in the regulation of genome flexibility. DNA methylation, accomplished by DNA methyltransferases, constitutes a prominent epigenetic modification of the chromatin fibre which is locked in a transcriptionally inactive conformation. Changes in DNA methylation accompany the reorganization of the nuclear architecture during plant cell differentiation and proliferation. After a stress treatment, in vitro-cultured microspores are reprogrammed and change their gametophytic developmental pathway towards embryogenesis, the process constituting a useful system of reprogramming in isolated cells for applied and basic research. Gene expression driven by developmental and stress cues often depends on DNA methylation; however, global DNA methylation and genome-wide expression patterns relationship is still poorly understood. In this work, the dynamics of DNA methylation patterns in relation to nuclear architecture and the expression of BnMET1a-like DNA methyltransferase genes have been analysed during pollen development and pollen reprogramming to embryogenesis in Brassica napus L. by a multidisciplinary approach. Results showed an epigenetic reprogramming after microspore embryogenesis induction which involved a decrease of global DNA methylation and its nuclear redistribution with the change of developmental programme and the activation of cell proliferation, while DNA methylation increases with pollen and embryo differentiation in a cell-type-specific manner. Changes in the presence, abundance, and distribution of BnMET1a-like transcripts highly correlated with variations in DNA methylation. Mature zygotic and pollen embryos presented analogous patterns of DNA methylation and MET1a-like expression, providing new evidence of the similarities between both developmental embryogenic programmes.
Testillano, Pilar S.
The molecular mechanisms underlying oogenesis and maternally controlled embryogenesis in fish are not fully understood, especially in marine species. Our aim was to study the egg and embryo transcriptome during oogenesis and early embryogenesis in Atlantic cod. Follicles from oogenesis stages (pre-, early-, and late-vitellogenic), ovulated eggs, and two embryonic stages (blastula, gastrula) were collected from broodstock fish and fertilized eggs. Gene expression profiles were measured in a 44?K oligo microarray consisting of 23,000 cod genes. Hundreds of differentially expressed genes (DEGs) were identified in the follicle stages investigated, implicating a continuous accumulation and degradation of polyadenylated transcripts throughout oogenesis. Very few DEGs were identified from ovulated egg to blastula, showing a more stable maternal RNA pool in early embryonic stages. The highest induction of expression was observed between blastula and gastrula, signifying the onset of zygotic transcription. During early vitellogenesis, several of the most upregulated genes are linked to nervous system signaling, suggesting increasing requirements for ovarian synaptic signaling to stimulate the rapid growth of oocytes. Highly upregulated genes during late vitellogenesis are linked to protein processing, fat metabolism, osmoregulation, and arrested meiosis. One of the genes with the highest upregulation in the ovulated egg is involved in oxidative phosphorylation, reflecting increased energy requirements during fertilization and the first rapid cell divisions of early embryogenesis. In conclusion, this study provides a large-scale presentation of the Atlantic cod's maternally controlled transcriptome in ovarian follicles through oogenesis, ovulated eggs, and early embryos. Mol. Reprod. Dev. 81: 619-635, 2014. © 2014 Wiley Periodicals, Inc. PMID:24687555
Kleppe, Lene; Edvardsen, Rolf Brudvik; Furmanek, Tomasz; Taranger, Geir Lasse; Wargelius, Anna
Stress-induced plant cell reprogramming involves changes in global genome organization, being the epigenetic modifications key factors in the regulation of genome flexibility. DNA methylation, accomplished by DNA methyltransferases, constitutes a prominent epigenetic modification of the chromatin fibre which is locked in a transcriptionally inactive conformation. Changes in DNA methylation accompany the reorganization of the nuclear architecture during plant cell differentiation and proliferation. After a stress treatment, in vitro-cultured microspores are reprogrammed and change their gametophytic developmental pathway towards embryogenesis, the process constituting a useful system of reprogramming in isolated cells for applied and basic research. Gene expression driven by developmental and stress cues often depends on DNA methylation; however, global DNA methylation and genome-wide expression patterns relationship is still poorly understood. In this work, the dynamics of DNA methylation patterns in relation to nuclear architecture and the expression of BnMET1a-like DNA methyltransferase genes have been analysed during pollen development and pollen reprogramming to embryogenesis in Brassica napus L. by a multidisciplinary approach. Results showed an epigenetic reprogramming after microspore embryogenesis induction which involved a decrease of global DNA methylation and its nuclear redistribution with the change of developmental programme and the activation of cell proliferation, while DNA methylation increases with pollen and embryo differentiation in a cell-type-specific manner. Changes in the presence, abundance, and distribution of BnMET1a-like transcripts highly correlated with variations in DNA methylation. Mature zygotic and pollen embryos presented analogous patterns of DNA methylation and MET1a-like expression, providing new evidence of the similarities between both developmental embryogenic programmes. PMID:23175669
Solís, María-Teresa; Rodríguez-Serrano, María; Meijón, Mónica; Cañal, María-Jesús; Cifuentes, Alejandro; Risueño, María C; Testillano, Pilar S
Somatic cell reprogramming is achieved by four reprogramming transcription factors (RTFs), Oct3/4, Sox2, Klf4, and c-Myc. However, in addition to the induction of pluripotent cells, these RTFs also generate pseudo-pluripotent cells, which do not show Nanog promoter activity. Therefore, it should be possible to fine-tune the RTFs to produce only fully pluripotent cells. For this study, a tagging system was developed to sort induced pluripotent stem (iPS) cells according to the expression levels of each of the four RTFs. Using this system, the most effective ratio (Oct3/4-high, Sox2-low, Klf4-high, c-Myc-high) of the RTFs was 88 times more efficient at producing iPS cells than the worst effective ratio (Oct3/4-low, Sox2-high, Klf4-low, c-Myc-low). Among the various RTF combinations, Oct3/4-high and Sox2-low produced the most efficient results. To investigate the molecular basis, microarray analysis was performed on iPS cells generated under high (Oct3/4-high and Sox2-low) and low (Oct3/4-low and Sox2-high) efficiency reprogramming conditions. Pathway analysis revealed that the G protein-coupled receptor (GPCR) pathway was up-regulated significantly under the high efficiency condition and treatment with the chemokine, C-C motif ligand 2, a member of the GPCR family, enhanced somatic cell reprogramming 12.3 times. Furthermore, data from the analysis of the signature gene expression profiles of mouse embryonic fibroblasts at 2 days after RTF infection revealed that the genetic modifier, Whsc1l1 (variant 1), also improved the efficiency of somatic cell reprogramming. Finally, comparison of the overall gene expression profiles between the high and low efficiency conditions will provide novel insights into mechanisms underlying somatic cell reprogramming.
Nagamatsu, Go; Saito, Shigeru; Kosaka, Takeo; Takubo, Keiyo; Kinoshita, Taisuke; Oya, Mototsugu; Horimoto, Katsuhisa; Suda, Toshio
Summary Conditions favourable to embryogenesis from isolated microspores of Brassica rapa L. ssp. oleifera (canola quality) were identified. A population with enhanced responsiveness for microspore embryogenesis (C200) was synthesized by crossing individual plants showing microspore embryogenic potential. For optimal microspore embryogenesis, buds (2–3mm in length, containing mid-late uninucieate microspores) were collected from older plants (2 months old) and microspores isolated and
Laurie Burnett; Stephen Yarrow; Bin Huang
Background Somatoform disorders are an important factor in functional disability and role impairment, though their independent contribution to disability has been unclear because of prevalent medical and psychiatric comorbidity. Objectives To assess the extent of the overlap of somatization with other psychiatric disorders and medical problems, to compare the functional disability and role impairment of somatizing and non-somatizing patients, and to determine the independent contribution of somatization to functional disability and role impairment. Design Patients were surveyed with self-report questionnaires assessing somatization, psychiatric disorder, and role impairment. Medical morbidity was indexed with a computerized medical record audit. Participants Consecutive adults making scheduled visits to their primary care physicians at two hospital-affiliated primary care practices on randomly chosen days. Measurements Intermediate activities of daily living, social activities, and occupational disability. Results Patients with somatization, as well as those with serious medical and psychiatric illnesses, had significantly more impairment of activities of daily life and social activities. When these predictors were considered simultaneously in a multivariable regression, the association with somatization remained highly significant and was comparable to or greater than many major medical conditions. Conclusions Patients with somatization had substantially greater functional disability and role impairment than non-somatizing patients. The degree of disability was equal to or greater than that associated with many major, chronic medical disorders. Adjusting the results for psychiatric and medical co-morbidity had little effect on these findings.
Orav, E. John; Bates, David W.; Barsky, Arthur J.
Cell suspension cultures were established from immature cotyledon derived calli from drought tolerant legume horsegram [Macrotyloma uniflorum (Lam.) Verdc.]. Embryogenic callus could be originated from cut slices of the immature cotyledons on MS solid medium [Murashige, T. Skoog, K., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497] augmented with 1.0?M zeatin
Shamsudeen Varisai Mohamed; Jih-Min Sung; Toong-Long Jeng; Chang-Sheng Wang
Carbenicillin, a well-known antibiotic, has been reported to have growth regulator-like activity in vitro for some plant species.\\u000a In the present paper we add horseradish (Armoracia rusticana) to the list of plants exhibiting such responses. This project began as an effort to eliminate latent bacterial contamination\\u000a among established in vitro horseradish plants. Carbenicillin (100 mg L?1) added to regeneration medium eliminated all
A. M. Shehata; W. Wannarat; R. M. Skirvin; M. A. Norton
The scarcity and stochastic nature of genetic mutations presents a significant challenge for scientists seeking to characterise\\u000a de novo mutation frequency at specific loci. Such mutations can be particularly numerous during regeneration of plants from\\u000a in vitro culture and can undermine the value of germplasm conservation efforts. We used cleaved amplified polymorphic sequence\\u000a (CAPS) analysis to characterise new mutations amongst
Carlos M. Rodríguez López; Hector Sicilia Bravo; Andrew C. Wetten; Michael J. Wilkinson
Eleutherococcus koreanum (E. koreanum) is an endangered medicinal plant, which has been used for the treatments of rheumatism, diabetes and hepatitis. An in vitro methodology has been developed for mass propagation of E. koreanum by using adventitious root explants in liquid cultures. Among the various strengths of Murashige and Skoog media sucrose and growth regulators tested, 1\\/3 strength hormone-free medium
So-Young Park; Jin-Kwon Ahn; Wi-Young Lee; Hosakatte Niranjana Murthy; Kee-Yoeup Paek
Summary Tissue cultures ofTriticum aestivum L. (wheat) initiated from young inflorescences and immature embryos possessed the potential for regeneration of whole plants. Both a friable and a compact type of callus were produced on Murashige and Skoog's medium with 2 mg\\/l 2,4-dichlorophenoxyacetic acid. The friable callus contained meristematic centers in which the peripheral cells ceased dividing, elongated, and could be
Peggy Ozias-Akins; Indra K. Vasil
The effect of different concentration of date palm syrup (2, 4, 6, 8 and 10%) and sucrose at concentration of 30 and 60 g\\/l in addition to the control (without carbon source) on the micro propagation of date palm \\
Symptoms of somatization were investigated in pediatric patients with recurrent abdominal pain (RAP) and comparison groups of patients with organic etiology for abdominal pain and well patients. Somatization scores were higher in RAP patients than well patients at the clinic visit, and higher than in either well patients or organic patients at a 3- month followup. Higher somatization scores in
Lynn S. Walker; Judy Garber; John W. Greene
Cannabis can have negative effects in its users, and a range of acute and chronic health problems associated with cannabis use has been dentified. Acute cannabis consumption is rarely lethal but it is associated with an increased risk of motor vehicle accident because of longer reaction time or impaired motor coordination. Chronic effects of cannabis use include generally cardiovascular and respiratory consequences but there are also oral, gastrointestinal, cutaneous and mucous, metabolic, gynecologic and obstetrical, sexual consequences, and cancer But associated tobacco smoking or other potential confounders may explain part of those somatic consequences. PMID:24579346
Cottencin, Olivier; Bence, Camille; Rolland, Benjamin; Karila, Laurent
Improvement of Hibiscus rosa-sinensis for increased frost tolerance has been attempted through somatic hybridization with the frost tolerant Lavatera thuringiaca. Cell suspensions from Hibiscus and Lavatera were transformed with A. tumefaciens harboring plasmids containing selectable genes coding for kanamycin and hygromycin resistance, respectively. We provided evidence that H. rosa-sinensis and L. thuringiaca were transformed by strong selection of transformed calluses in medium containing antibiotics, by GUS activity determination in protein extracts and by molecular confirmation of chromosomal integration and expression of the selectable genes. Protoplasts isolated from a kanamycinresistant Hibiscus callus and from a hygromycin-resistant Lavatera callus were fused and selected in medium containing both antibiotics. We determined unambiguously that the regenerated double-antibiotic resistant clones obtained are indeed somatic hybrids through analysis of acid phosphatase zymograms and nuclear DNA content. Plant regeneration through somatic embryogenesis was accomplished from both isolated protoplasts and transgenic calluses of L. thuringiaca. However, regeneration from the double-antibiotic resistant fusant calluses was unsuccessful. Analysis of the somatic hybrids at the callus level showed that chilling and freezing tolerance are governed by independent genetic components. The somatic hybrids displayed significant improvement for chilling tolerance at conditions lethal to H. rosa-sinensis, although frost tolerance was not expressed. PMID:24178462
Vazquez-Thello, A; Li Yang, J; Hidaka, M; Uozumi, T
Somatic hybridizations via electrofusion were performed in combinations of Oriental hybrid lilies (cvs. Acapulco and Shirotae) and Liliumxformolongi hort. (cv. Hakucho). Electrofusion-treated protoplasts divided only under nurse culture. The divided protoplasts grew into calli on the culture medium containing picloram, and the calli were then transferred to the hormone-free culture medium for induction of plant regeneration. The regenerants were transferred to a greenhouse, and were grown until the flower stage. In the fusion combinations of Acapulco + Hakucho and Shirotae + Hakucho, four regenerants apparently showed different morphological features compared with their parents. The results of molecular analyses by means of cleaved amplified polymorphic sequences markers and flow cytometry confirmed that these regenerants were somatic hybrid plants. Furthermore, we examined the stability of the morphological features of the hybrids in the next generations. This is the first report to describe the successful realization of Lilium somatic hybridization via protoplast fusion. PMID:12684784
Horita, Mitsugu; Morohashi, Hitomi; Komai, Fuminori
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.
Abbott, M. K.; Spooner, B. S. (Principal Investigator)
BACKGROUND: The consecutive acquisition of genetic alterations characterizes neoplastic processes. As a consequence of these alterations, molecular interactions are reprogrammed in the context of highly connected and regulated cellular networks. The recent identification of the collection of somatically mutated genes in breast tumors (breast cancer somatic \\
Pilar Hernández; Xavier Solé; Joan Valls; Víctor Moreno; Gabriel Capellá; Ander Urruticoechea; Miguel Angel Pujana
The authors review the research on childhood antecedents and personality contributions to the somatoform disorders, as well as research on social influences during adulthood. Based on these data, the authors hypothesize that somatizing patients display anxious attachment behavior that derives from childhood experiences with caregivers. Early exposure to illness increases the likeli- hood that distress will be manifested somatically. When
SCOTT STUART; RUSSELL NOYES
Six identical bioreactors were constructed and built at the Agricultural University of Norway to provide optimal conditions for plant cell regeneration from cells into somatic embryos (‘clonal or somatic seeds’). This was made possible through cooperation in COST87 by a European network in a working group on regeneration from plant cell cultures. The bioreactor design provides gentle stirring through a
Anne Kathrine Hvoslef-Eide; Odd Arild S. Olsen; Ragnhild Lyngved; Cristel Munster; Petter H. Heyerdahl
Six identical bioreactors were constructed and built at the Agricultural University of Norway to provide optimal conditions for plant cell regeneration from cells into somatic embryos (“clonal or somatic seeds”). This was made possible through cooperation in COST87 by a European network in a working group on regeneration from plant cell cultures. The bioreactor design provides gentle stirring through a
Anne Kathrine Hvoslef-Eide; Odd Olsen; Ragnhild Lyngved; Cristel Munster; Petter Heyerdahl
Long noncoding RNAs (lncRNAs) comprise a diverse class of transcripts that structurally resemble mRNAs but do not encode proteins. Recent genome-wide studies in humans and the mouse have annotated lncRNAs expressed in cell lines and adult tissues, but a systematic analysis of lncRNAs expressed during vertebrate embryogenesis has been elusive. To identify lncRNAs with potential functions in vertebrate embryogenesis, we performed a time-series of RNA-seq experiments at eight stages during early zebrafish development. We reconstructed 56,535 high-confidence transcripts in 28,912 loci, recovering the vast majority of expressed RefSeq transcripts while identifying thousands of novel isoforms and expressed loci. We defined a stringent set of 1133 noncoding multi-exonic transcripts expressed during embryogenesis. These include long intergenic ncRNAs (lincRNAs), intronic overlapping lncRNAs, exonic antisense overlapping lncRNAs, and precursors for small RNAs (sRNAs). Zebrafish lncRNAs share many of the characteristics of their mammalian counterparts: relatively short length, low exon number, low expression, and conservation levels comparable to that of introns. Subsets of lncRNAs carry chromatin signatures characteristic of genes with developmental functions. The temporal expression profile of lncRNAs revealed two novel properties: lncRNAs are expressed in narrower time windows than are protein-coding genes and are specifically enriched in early-stage embryos. In addition, several lncRNAs show tissue-specific expression and distinct subcellular localization patterns. Integrative computational analyses associated individual lncRNAs with specific pathways and functions, ranging from cell cycle regulation to morphogenesis. Our study provides the first systematic identification of lncRNAs in a vertebrate embryo and forms the foundation for future genetic, genomic, and evolutionary studies.
Pauli, Andrea; Valen, Eivind; Lin, Michael F.; Garber, Manuel; Vastenhouw, Nadine L.; Levin, Joshua Z.; Fan, Lin; Sandelin, Albin; Rinn, John L.; Regev, Aviv; Schier, Alexander F.
During fruit fly embryogenesis, there are several morphogenetic events in which sheets of epithelial cells expand, contract and bend due to coordinated intra- and intercellular forces. This tissue-level reshaping is accompanied by changes in the shape and arrangement of individual cells -- changes that can be measured quantitatively and dynamically using modern live-cell imaging techniques. Such data sets represent rich targets for computational modeling of self-organization; however, reproducing the observed cell- and tissue-level reshaping is not enough. The inverse problem of using cell shape changes to determine cell-level forces is ill-posed -- yielding non-unique solutions that cannot discriminate between active changes in cell shape and passive deformation. These non-unique solutions can be tested experimentally using in vivo laser-microsurgery -- i.e., cutting a targeted region of an epithelium and carefully tracking the temporal and spatial dependence of the subsequent strain relaxation. This technique uses a variety of incisions (hole, line or closed curve) to probe different aspects of epithelial mechanics: the local mesoscopic strain; the distribution of intracellular forces; changes in the cell-level power-law rheology; and the question of active versus passive deformation. I will discuss my group's work using laser-microsurgery to investigate two morphogenetic events in fruit fly embryogenesis: germband retraction and dorsal closure. In both cases, we find a substantial active mechanical role for the amnioserosa -- an epithelium that undergoes apoptosis near the end of embryogenesis and makes no part of the fly larva -- in reshaping an adjacent epithelium that becomes the larval epidermis. In these examples, self-organization of the fly embryo relies not only on self-organization of individual tissues, but also on the mechanical interactions between tissues.
Hutson, M. Shane
We have cloned and characterized a second member of the Xenopus CAP (cyclase associated protein) gene family. xCAP2 demonstrates greater restriction of expression than its homolog, xCAP1, and is differentially expressed throughout early embryogenesis. Although present as a maternal transcript, CAP2 comes to be expressed in the anterior-most mesoderm/endoderm during late gastrulation, in paraxial mesoderm during late neurula stages, and later expresses in lens, cardiac primordia, somites, otic vesicles, retina, and in the optic and craniofacial musculature. The gene is also expressed in the leading edge of myotome. PMID:19598124
Wolanski, Marian; Khosrowshahian, Farhad; Jerant, Lara; Jap, Ing-Suan; Brockman, Jennifer; Crawford, Michael J
Plant hemoglobins are ubiquitous molecules involved in several aspects of plant development and stress responses. Studies on the functional aspects of plant hemoglobins at the cellular level in these processes are limited, despite their ability to scavenge nitric oxide (NO), an important signal molecule interfering with hormone synthesis and sensitivity. This mini-review summarizes current knowledge on plant hemoglobins, analyzes their participation in plant pathogen interaction and embryogenesis and proposes a possible model centering on jasmonic acid (JA) as a downstream component of hemoglobin responses.
Wally, Owen S.D.; Mira, Mohamed M.; Hill, Robert D.; Stasolla, Claudio
Factors which influence somatic cell counts in bovine milk are reviewed and guidelines for their interpretation are presented. It is suggested that the thresholds of 300 000 and 250 000 cells/mL be used to identify infected quarters and cows respectively. However, it is stressed that somatic cell counts are general indicators of udder health which are subject to the influence of many factors. Therefore the evaluation of several successive counts is preferable to the interpretation of an individual count. Relationships between somatic cell counts and both milk production and milk composition are discussed. Subclinical mastitis reduces milk quality and decreases yield although the relationship between production loss and somatic cell count requires clarification. Finally the availability of somatic cell counting programs in Canada is presented.
Dohoo, I. R.; Meek, A. H.
In earlier studies, only two major patterns of transcript accumulation were seen for 18 late embryogenesis abundant (Lea) gene families in cotton (Gossypium hirsutum L.) during embryogenesis and early germination. Each of these gene families probably comprises two active alloalleles. The two polypeptides encoded by seven of the Lea families can be distinguished, and analysis of their translation in vitro
D. Wayne Hughes; Glenn A. Galau
Somatic treatments for mood disorders represent a class of interventions available either as a stand-alone option, or in combination with psychopharmacology and/or psychotherapy. Here, we review the currently available techniques, including those already in clinical use and those still under research. Techniques are grouped into the following categories: (1) seizure therapies, including electroconvulsive therapy and magnetic seizure therapy, (2) noninvasive techniques, including repetitive transcranial magnetic stimulation, transcranial direct current stimulation, and cranial electric stimulation, (3) surgical approaches, including vagus nerve stimulation, epidural electrical stimulation, and deep brain stimulation, and (4) technologies on the horizon. Additionally, we discuss novel approaches to the optimization of each treatment, and new techniques that are under active investigation. PMID:21976043
Rosa, Moacyr A; Lisanby, Sarah H
Bone morphogenetic protein 15 (BMP15) belongs to an unusual subgroup of the transforming growth factor ? (TGF?) superfamily of signaling ligands as it lacks a key cysteine residue in the mature region required for proper intermolecular dimerization. Naturally occurring BMP15 mutation leads to early ovarian failure in humans, and BMP15 has been shown to activate the Smad1/5/8 pathway in that context. Despite its important role in germ cell specification, the embryological function of BMP15 remains unknown. Surprisingly, we find that during early Xenopus embryogenesis BMP15 acts solely as an inhibitor of the Smad1/5/8 pathway and the Wnt pathway. BMP15 gain-of-function leads to embryos with secondary ectopic heads and to direct neural induction in intact explants. BMP15 inhibits BMP4-mediated epidermal induction in dissociated explants. BMP15 strongly inhibits BRE response induced by BMP4 and blocks phosphorylation and activation of Smad1/5/8 MH2-domain. Mechanistically, BMP15 protein specifically interacts with BMP4 protein, suggesting inhibition upstream of receptor binding. Loss-of-function experiments using morpholinos or a naturally occurring human BMP15 dominant-negative mutant (BMP15-Y235C) leads to embryos lacking head. BMP15-Y235C also eliminates the inhibitory activity of BMP15 on BRE (BMP-responsive element). Finally, we show that BMP15 inhibits the canonical branch of the Wnt pathway, upstream of ?-catenin. We, thus, demonstrate that BMP15 is necessary and sufficient for the specification of dorso-anterior structures and highlight novel mechanisms of BMP15 function that strongly suggest a reinterpretation of its function in ovaries specially for ovarian failure.
Di Pasquale, Elisa; Brivanlou, Ali H.
Epidemiological studies have correlated arsenic exposure in drinking water with adverse developmental outcomes such as stillbirths, spontaneous abortions, neonatal mortality, low birth weight, delays in the use of musculature, and altered locomotor activity. Killifish (Fundulus heteroclitus) were used as a model to help to determine the mechanisms by which arsenic could impact development. Killifish embryos were exposed to three different sodium arsenite concentrations and were collected at 32 h post-fertilization (hpf), 42 hpf, 168 hpf, or < 24 h post-hatch. A killifish oligo microarray was developed and used to examine gene expression changes between control and 25-ppm arsenic-exposed hatchlings. With artificial neural network analysis of the transcriptomic data, accurate prediction of each group (control vs. arsenic-exposed embryos) was obtained using a small subset of only 332 genes. The genes differentially expressed include those involved in cell cycle, development, ubiquitination, and the musculature. Several of the genes involved in cell cycle regulation and muscle formation, such as fetuin B, cyclin D-binding protein 1, and CapZ, were differentially expressed in the embryos in a time- and dose-dependent manner. Examining muscle structure in the hatchlings showed that arsenic exposure during embryogenesis significantly reduces the average muscle fiber size, which is coupled with a significant 2.1- and 1.6-fold upregulation of skeletal myosin light and heavy chains, respectively. These findings collectively indicate that arsenic exposure during embryogenesis can initiate molecular changes that appear to lead to aberrant muscle formation. PMID:22058191
Gaworecki, Kristen M; Chapman, Robert W; Neely, Marion G; D'Amico, Angela R; Bain, Lisa J
Somatic fixation occurs when the patient or physician focuses exclusively on the somatic aspects of a complex problem. This common and challenging problem results from individual, family, and cultural factors that promote communication and the expression of emotional experience through somatic symptoms. An unrewarding cycle of interactions occurs when the physician first rules out organic illness in the somatically fixated
Susan H. McDaniel; Thomas Campbell; David Seaburn
Evolution is based on genetic variability and subsequent phenotypic selection. Mechanisms that modulate the rate of mutation according to environmental cues, and thus control the balance between genetic stability and flexibility, might provide a distinct evolutionary advantage. Stress-induced mutations stimulated by unfavorable environments, and possible mechanisms for their induction, have been described for several organisms, but research in this area has mainly focused on microorganisms. We have analyzed the influence of adverse environmental conditions on the genetic stability of the higher plant Arabidopsis thaliana. Here we show that a biotic stress factor-attack by the oomycete pathogen Peronospora parasitica-can stimulate somatic recombination in Arabidopsis. The same effect was observed when plant pathogen-defense mechanisms were activated by the chemicals 2,6-dichloroisonicotinic acid (INA) or benzothiadiazole (BTH), or by a mutation (cim3). Together with previous studies of recombination induced by abiotic factors, these findings suggest that increased somatic recombination is a general stress response in plants. The increased genetic flexibility might facilitate evolutionary adaptation of plant populations to stressful environments. PMID:11836502
Lucht, Jan M; Mauch-Mani, Brigitte; Steiner, Henry-York; Metraux, Jean-Pierre; Ryals, John; Hohn, Barbara
Cancer evolves by mutation, with somatic reactivation of retrotransposons being one such mutational process. Germline retrotransposition can cause processed pseudogenes, but whether this occurs somatically has not been evaluated. Here we screen sequencing data from 660 cancer samples for somatically acquired pseudogenes. We find 42 events in 17 samples, especially non-small cell lung cancer (5/27) and colorectal cancer (2/11). Genomic features mirror those of germline LINE element retrotranspositions, with frequent target-site duplications (67%), consensus TTTTAA sites at insertion points, inverted rearrangements (21%), 5' truncation (74%) and polyA tails (88%). Transcriptional consequences include expression of pseudogenes from UTRs or introns of target genes. In addition, a somatic pseudogene that integrated into the promoter and first exon of the tumour suppressor gene, MGA, abrogated expression from that allele. Thus, formation of processed pseudogenes represents a new class of mutation occurring during cancer development, with potentially diverse functional consequences depending on genomic context. PMID:24714652
Cooke, Susanna L; Shlien, Adam; Marshall, John; Pipinikas, Christodoulos P; Martincorena, Inigo; Tubio, Jose M C; Li, Yilong; Menzies, Andrew; Mudie, Laura; Ramakrishna, Manasa; Yates, Lucy; Davies, Helen; Bolli, Niccolo; Bignell, Graham R; Tarpey, Patrick S; Behjati, Sam; Nik-Zainal, Serena; Papaemmanuil, Elli; Teixeira, Vitor H; Raine, Keiran; O'Meara, Sarah; Dodoran, Maryam S; Teague, Jon W; Butler, Adam P; Iacobuzio-Donahue, Christine; Santarius, Thomas; Grundy, Richard G; Malkin, David; Greaves, Mel; Munshi, Nikhil; Flanagan, Adrienne M; Bowtell, David; Martin, Sancha; Larsimont, Denis; Reis-Filho, Jorge S; Boussioutas, Alex; Taylor, Jack A; Hayes, Neil D; Janes, Sam M; Futreal, P Andrew; Stratton, Michael R; McDermott, Ultan; Campbell, Peter J
Cancer evolves by mutation, with somatic reactivation of retrotransposons being one such mutational process. Germline retrotransposition can cause processed pseudogenes, but whether this occurs somatically has not been evaluated. Here we screen sequencing data from 660 cancer samples for somatically acquired pseudogenes. We find 42 events in 17 samples, especially non-small cell lung cancer (5/27) and colorectal cancer (2/11). Genomic features mirror those of germline LINE element retrotranspositions, with frequent target-site duplications (67%), consensus TTTTAA sites at insertion points, inverted rearrangements (21%), 5? truncation (74%) and polyA tails (88%). Transcriptional consequences include expression of pseudogenes from UTRs or introns of target genes. In addition, a somatic pseudogene that integrated into the promoter and first exon of the tumour suppressor gene, MGA, abrogated expression from that allele. Thus, formation of processed pseudogenes represents a new class of mutation occurring during cancer development, with potentially diverse functional consequences depending on genomic context.
Cooke, Susanna L.; Shlien, Adam; Marshall, John; Pipinikas, Christodoulos P.; Martincorena, Inigo; Tubio, Jose M.C.; Li, Yilong; Menzies, Andrew; Mudie, Laura; Ramakrishna, Manasa; Yates, Lucy; Davies, Helen; Bolli, Niccolo; Bignell, Graham R.; Tarpey, Patrick S.; Behjati, Sam; Nik-Zainal, Serena; Papaemmanuil, Elli; Teixeira, Vitor H.; Raine, Keiran; O'Meara, Sarah; Dodoran, Maryam S.; Teague, Jon W.; Butler, Adam P.; Iacobuzio-Donahue, Christine; Santarius, Thomas; Grundy, Richard G.; Malkin, David; Greaves, Mel; Munshi, Nikhil; Flanagan, Adrienne M.; Bowtell, David; Martin, Sancha; Larsimont, Denis; Reis-Filho, Jorge S.; Boussioutas, Alex; Taylor, Jack A.; Hayes, Neil D.; Janes, Sam M.; Futreal, P. Andrew; Stratton, Michael R.; McDermott, Ultan; Campbell, Peter J.; Provenzano, Elena; van de Vijver, Marc; Richardson, Andrea L.; Purdie, Colin; Pinder, Sarah; Mac Grogan, Gaetan; Vincent-Salomon, Anne; Larsimont, Denis; Grabau, Dorthe; Sauer, Torill; Garred, Øystein; Ehinger, Anna; Van den Eynden, Gert G.; van Deurzen, C.H.M; Salgado, Roberto; Brock, Jane E.; Lakhani, Sunil R.; Giri, Dilip D.; Arnould, Laurent; Jacquemier, Jocelyne; Treilleux, Isabelle; Caldas, Carlos; Chin, Suet-Feung; Fatima, Aquila; Thompson, Alastair M.; Stenhouse, Alasdair; Foekens, John; Martens, John; Sieuwerts, Anieta; Brinkman, Arjen; Stunnenberg, Henk; Span, Paul N.; Sweep, Fred; Desmedt, Christine; Sotiriou, Christos; Thomas, Gilles; Broeks, Annegein; Langerod, Anita; Aparicio, Samuel; Simpson, Peter T.; van ’t Veer, Laura; Erla Eyfjörd, Jórunn; Hilmarsdottir, Holmfridur; Jonasson, Jon G.; Børresen-Dale, Anne-Lise; Lee, Ming Ta Michael; Wong, Bernice Huimin; Tan, Benita Kiat Tee; Hooijer, Gerrit K.J.
The principle of linear induction acceleration is described, and examples are given of practical configurations for induction linacs. These examples include the Advanced Technology Accelerator, Long Pulse Induction Linac, Radial Line Accelerator (RADLAC), and Magnetically-Insulated Electron-Focussed Ion Linac. A related concept, the auto accelerator, is described in which the high-current electron-beam technology in the sub-10 MeV region is exploited to produce electron beams at energies perhaps as high as the 100 to 1000 MeV range. Induction linacs for ions are also discussed. The efficiency of induction linear acceleration is analyzed. (LEW)
The somatic marker hypothesis (SMH; [Damasio, A. R., Tranel, D., Damasio, H., 1991. Somatic markers and the guidance of behaviour: theory and preliminary testing. In Levin, H.S., Eisenberg, H.M., Benton, A.L. (Eds.), Frontal Lobe Function and Dysfunction. Oxford University Press, New York, pp. 217–229]) proposes that emotion-based biasing signals arising from the body are integrated in higher brain regions, in
Barnaby D. Dunn; Tim Dalgleish; Andrew D. Lawrence
This document presents arguments that conclude that it is unethical to use somatic cell nuclear transfer (SCNT) for infertility treatment due to concerns about safety; the unknown impact of SCNT on children, families, and society; and the availability of other ethically acceptable means of assisted reproduction. This document replaces the ASRM Ethics Committee report titled, "Human somatic cell nuclear transfer (cloning)," last published in Fertil Steril 2000;74:873-6. PMID:22795681
This study examined (a) demographic and clinical characteristics associated with physical symptoms in anxiety-disordered youth and (b) the impact of cognitive-behavioral therapy (Coping Cat), medication (sertraline), their combination, and pill placebo on physical symptoms. Youth (N = 488, ages 7-17 years) with a principal diagnosis of generalized anxiety disorder, separation anxiety disorder, or social phobia participated as part of a multi-site, randomized controlled trial and received treatment delivered over 12 weeks. Diagnostic status, symptom severity, and impairment were assessed at baseline and week 12. The total number and severity of physical symptoms was associated with age, principal diagnosis, anxiety severity, impairment, and the presence of comorbid internalizing disorders. Common somatic complaints were headaches, stomachaches, head cold or sniffles, sleeplessness, and feeling drowsy or too sleepy. Physical symptoms decreased over the course of treatment, and were unrelated to treatment condition. Clinical implications and directions for future research are discussed (ClinicalTrials.gov number, NCT00052078). PMID:24129543
Crawley, Sarah A; Caporino, Nicole E; Birmaher, Boris; Ginsburg, Golda; Piacentini, John; Albano, Anne Marie; Sherrill, Joel; Sakolsky, Dara; Compton, Scott N; Rynn, Moira; McCracken, James; Gosch, Elizabeth; Keeton, Courtney; March, John; Walkup, John T; Kendall, Philip C
We studied the effect of three drilling muds on the early developmental stages of the rainbow trout. The following parameters were examined in the experiments: embryo mortality rate, rate of development, time of the beginning and end of individual stages and the duration embryogenesis on the whole, presence of developmental abnormalities, timing of larval hatching and morphometric indices of the larvae. Minimal inhibitory concentrations of the drilling muds were determined. The gel-humate drilling mud was the most toxic. At its level equal to 0.1-5.0 g/liter significant dose-dependent inhibition of their linear growth rate and the rate of weight increase, was found despite the high survival rate of the larvae. 4 refs., 7 tabs.
Kosheleva, V.V.; Novikov, M.A.; Migalovskii, I.P.
Nucleosome organization at promoter regions plays an important role in regulating gene activity. Genome-wide studies in yeast, flies, worms, mammalian embryonic stem cells and transformed cell lines have found well-positioned nucleosomes flanking a nucleosome depleted region (NDR) at transcription start sites. This nucleosome arrangement depends on DNA sequence (cis-elements) as well as DNA binding factors and ATP-dependent chromatin modifiers (trans-factors). However, little is understood about how the nascent embryonic genome positions nucleosomes during development. This is particularly intriguing since the embryonic genome must undergo a broad reprogramming event upon fusion of sperm and oocyte. Using four stages of early embryonic zebrafish development, we map nucleosome positions at the promoter region of 37 zebrafish hox genes. We find that nucleosome arrangement at the hox promoters is a progressive process that takes place over several stages. At stages immediately after fertilization, nucleosomes appear to be largely disordered at hox promoter regions. At stages after activation of the embryonic genome, nucleosomes are detectable at hox promoters, with positions becoming more uniform and more highly occupied. Since the genomic sequence is invariant during embryogenesis, this progressive change in nucleosome arrangement suggests that trans-factors play an important role in organizing nucleosomes during embryogenesis. Separating hox genes into expressed and non-expressed groups shows that expressed promoters have better positioned and occupied nucleosomes, as well as distinct NDRs, than non-expressed promoters. Finally, by blocking the retinoic acid-signaling pathway, we disrupt early hox gene transcription, but observe no effect on nucleosome positions, suggesting that active hox transcription is not a driving force behind the arrangement of nucleosomes at the promoters of hox genes during early development.
Weicksel, Steven E.; Xu, Jia; Sagerstrom, Charles G.
As we previously reported for glutathione (GSH), both ascorbic acid (AA) and vitamin E were observed to suppress wild carrot (Daucus carota L.) somatic embryogenesis with little concomitant effect on biomass. Endogenous concentrations of AA were lower during embryo development than during cell proliferation, exhibiting a temporal pattern nearly identical to that of GSH. GSSG (oxidized GSH) reductase was found to be considerably more active in proliferating than in developing cultures, whereas no difference was evident in the case of dehydroascorbate (DHA) reductase. Both GSH and AA concentrations in these cells are governed by 2,4-D. These results show that redox status is a strong determinant of proliferative versus developmental growth and indicate that the mode of action of 2,4-D in this system may be explained at least in part by its influence on endogenous antioxidant levels. PMID:16665669
Earnshaw, B A; Johnson, M A
Pollen embryogenesis provides exciting opportunities in the areas of breeding and biotechnology as well as representing a convenient model for studying the process of plant cell proliferation in general and embryogenesis in particular. A cell culture system was devised in which immature barley pollen could be cultured as a monolayer trapped between the bottom glass-cover slip of a live-cell chamber and a diaphanous PTFE membrane within a liquid medium over a period of up to 28 d, allowing the process of embryogenesis to be tracked in individual pollen. Z-stacks of images were automatically captured every 3min, starting from the unicellular pollen stage up to the development of multicellular, embryogenic structures. The method should prove useful for the elucidation of ultrastructural features and molecular processes associated with pollen embryogenesis.
The importance of chromosome studies during successive stages of embryogenesis for understanding the genetic mechanisms of individual development is emphasized. Methods are given for chromosome analysis in all stages of male and female gametogenesis and s...
A. P. Dyban V. S. Baranov
Arabidopsis thaliana seeds without functional SEED MATURATION PROTEIN1 (SMP1), a boiling soluble protein predicted to be of intrinsic disorder, presumed to be a LATE EMBRYOGENESIS ABUNDANT (LEA) family protein based on sequence homology, do not enter secondary dormancy after 3 days at 40 °C. We hypothesized that SMP1 may protect a heat labile protein involved in the promotion of secondary dormancy. Recombinant SMP1 and GmPM28, its soybean (Glycine max), LEA4 homologue, protected the labile GLUCOSE-6-PHOSPHATE DEHYROGENASE enzyme from heat stress, as did a known protectant, Bovine Serum Albumin, whether the LEA protein was in solution or attached to the bottom of microtiter plates. Maintenance of a biological function for both recombinant LEA proteins when immobilized encouraged a biopanning approach to screen for potential protein interactors. Phage display with two Arabidopsis seed, T7 phage, cDNA libraries, normalized for transcripts present in the mature, dehydrated, 12-, 24-, or 36-h imbibed seeds, were used in biopans against recombinant SMP1 and GmPM28. Phage titer increased considerably over four rounds of biopanning for both LEA proteins, but not for BSA, at both 25 and at 41 °C, regardless of the library used. The prevalence of multiple, independent clones encoding portions of specific proteins repeatedly retrieved from different libraries, temperatures and baits, provides evidence suggesting these LEA proteins are discriminating which proteins they protect, a novel finding. The identification of putative LEA-interacting proteins provides targets for reverse genetic approaches to further dissect the induction of secondary dormancy in seeds in response to heat stress. PMID:22837651
Kushwaha, Rekha; Lloyd, Taylor D; Schäfermeyer, Kim R; Kumar, Santosh; Downie, Allan Bruce
Using callus derived from immature embryos, regeneration of viable plants was obtained in soybean (Glycine max (L.) Merr.). Depending on the composition of the medium, regeneration occurred via embryogenesis or via organogenesis. Embryogenesis resulted when embryos were plated on Murashige and Skoog (MS) medium containing 43 µM a-naphthaleneacetic acid. In work with the cultivar Williams 82, the addition of 5.0
U. B. Barwale; H. R. Kerns; J. M. Widholm
Differentiating true somatic mutations from artifacts in massively parallel sequencing data is an immense challenge. To develop methods for optimal somatic mutation detection and to identify factors influencing somatic mutation prediction accuracy, we validated predictions from three somatic mutation detection algorithms, MuTect, JointSNVMix2 and SomaticSniper, by Sanger sequencing. Full consensus predictions had a validation rate of >98%, but some partial consensus predictions validated too. In cases of partial consensus, read depth and mapping quality data, along with additional prediction methods, aided in removing inaccurate predictions. Our consensus approach is fast, flexible and provides a high-confidence list of putative somatic mutations.
Somatic hypermutation of Ig genes is initiated by transcription-coupled cytidine deamination in Ig loci. Error-prone processing of the resultant DNA lesions is thought to cause extensive mutagenesis, but it is presently an enigma how and why error-prone rather than error-free repair pathways are recruited. During DNA replication, recruitment of error-prone translesion polymerases may be mediated by Rad6/Rad18-mediated ubiquitination of proliferating cell nuclear antigen, a major switchboard controlling the fidelity of DNA lesion bypass in eukaryotes. By inactivation of Rad18 in the DT40 B cell line, we show that the Rad6 pathway is involved in somatic hypermutation in these cells. Our findings imply that targeted recruitment of mutagenic polymerases by the Rad6 pathway contributes to the complex process of somatic hypermutation and provide a framework for more detailed mechanistic studies of the mutagenesis phase of secondary Ig diversification.
Bachl, Jurgen; Ertongur, Isin; Jungnickel, Berit
Tissue homeostasis and regenerative capacity rely on rare populations of somatic stem cells endowed with the potential to self-renew and differentiate. During aging, many tissues show a decline in regenerative potential coupled with a loss of stem cell function. Cells including somatic stem cells have evolved a series of checks and balances to sense and repair cellular damage to maximize tissue function. However, during aging the mechanisms that protect normal cell function begin to fail. In this review, we will discuss how common cellular mechanisms that maintain tissue fidelity and organismal lifespan impact somatic stem cell function. We will highlight context-dependent changes and commonalities that define aging, by focusing on three age-sensitive stem cell compartments: blood, neural, and muscle. Understanding the interaction between extrinsic regulators and intrinsic effectors that operate within different stem cell compartments is likely to have important implications for identifying strategies to improve health span and treat age-related degenerative diseases.
Mitogen-activated protein kinase (MAPK) is activated by MAPK kinase (MAPKK) in a variety of signaling pathways. This kinase cascade has been shown to function in cell proliferation and differentiation, but its role in early vertebrate development remains to be investigated. During early vertebrate embryogenesis, the induction and patterning of mesoderm are thought to be determined by signals from intercellular factors such as members of the fibroblast growth factor (FGF) family and members of the transforming growth factor-beta family. Here we show that the microinjection of either mRNA encoding a constitutively active mutant of MAPKK or mRNA encoding a constitutively active form of STE11, a MAPKK kinase, leads to the induction of mesoderm in ectodermal explants from Xenopus embryos. Moreover, the expression of MAPK phosphatase-1 (MKP-1, also called CL100) blocks the growth factor-stimulated mesoderm induction. Furthermore, injection of CL100 mRNA into two-cell stage embryos causes severe defects in gastrulation and posterior development. The effects induced by CL100 can be rescued by co-injection of wild-type MAPK mRNA. Thus, the MAPK cascade may play a crucial role in early vertebrate embryogenesis, especially during mesoderm induction. Images
Gotoh, Y; Masuyama, N; Suzuki, A; Ueno, N; Nishida, E
Background Many aspects of autoimmune disease are not well understood, including the specificities of autoimmune targets, and patterns of co-morbidity and cross-heritability across diseases. Prior work has provided evidence that somatic mutation caused by gene conversion and deletion at segmentally duplicated loci is relevant to several diseases. Simple tandem repeat (STR) sequence is highly mutable, both somatically and in the germ-line, and somatic STR mutations are observed under inflammation. Results Protein-coding genes spanning STRs having markers of mutability, including germ-line variability, high total length, repeat count and/or repeat similarity, are evaluated in the context of autoimmunity. For the initiation of autoimmune disease, antigens whose autoantibodies are the first observed in a disease, termed primary autoantigens, are informative. Three primary autoantigens, thyroid peroxidase (TPO), phogrin (PTPRN2) and filaggrin (FLG), include STRs that are among the eleven longest STRs spanned by protein-coding genes. This association of primary autoantigens with long STR sequence is highly significant (). Long STRs occur within twenty genes that are associated with sixteen common autoimmune diseases and atherosclerosis. The repeat within the TTC34 gene is an outlier in terms of length and a link with systemic lupus erythematosus is proposed. Conclusions The results support the hypothesis that many autoimmune diseases are triggered by immune responses to proteins whose DNA sequence mutates somatically in a coherent, consistent fashion. Other autoimmune diseases may be caused by coherent somatic mutations in immune cells. The coherent somatic mutation hypothesis has the potential to be a comprehensive explanation for the initiation of many autoimmune diseases.
Ross, Kenneth Andrew
Er71 mutant embryos are nonviable and lack hematopoietic and endothelial lineages. To further define the functional role for ER71 in cell lineage decisions, we generated genetically modified mouse models. We engineered an Er71-EYFP transgenic mouse model by fusing the 3.9 kb Er71 promoter to the EYFP reporter gene. Using FACS and transcriptional profiling, we examined the EYFP(+) population of cells in Er71 mutant and wild-type littermates. In the absence of ER71, we observed an increase in the number of EYFP-expressing cells, increased expression of the cardiac molecular program and decreased expression of the hemato-endothelial program, as compared with wild-type littermate controls. We also generated a novel Er71-Cre transgenic mouse model using the same 3.9 kb Er71 promoter. Genetic fate-mapping studies revealed that the ER71-expressing cells give rise to the hematopoietic and endothelial lineages in the wild-type background. In the absence of ER71, these cell populations contributed to alternative mesodermal lineages, including the cardiac lineage. To extend these analyses, we used an inducible embryonic stem/embryoid body system and observed that ER71 overexpression repressed cardiogenesis. Together, these studies identify ER71 as a critical regulator of mesodermal fate decisions that acts to specify the hematopoietic and endothelial lineages at the expense of cardiac lineages. This enhances our understanding of the mechanisms that govern mesodermal fate decisions early during embryogenesis. PMID:21989919
Rasmussen, Tara L; Kweon, Junghun; Diekmann, Mackenzie A; Belema-Bedada, Fikru; Song, Qingfeng; Bowlin, Kathy; Shi, Xiaozhong; Ferdous, Anwarul; Li, Tongbin; Kyba, Michael; Metzger, Joseph M; Koyano-Nakagawa, Naoko; Garry, Daniel J
Er71 mutant embryos are nonviable and lack hematopoietic and endothelial lineages. To further define the functional role for ER71 in cell lineage decisions, we generated genetically modified mouse models. We engineered an Er71-EYFP transgenic mouse model by fusing the 3.9 kb Er71 promoter to the EYFP reporter gene. Using FACS and transcriptional profiling, we examined the EYFP+ population of cells in Er71 mutant and wild-type littermates. In the absence of ER71, we observed an increase in the number of EYFP-expressing cells, increased expression of the cardiac molecular program and decreased expression of the hemato-endothelial program, as compared with wild-type littermate controls. We also generated a novel Er71-Cre transgenic mouse model using the same 3.9 kb Er71 promoter. Genetic fate-mapping studies revealed that the ER71-expressing cells give rise to the hematopoietic and endothelial lineages in the wild-type background. In the absence of ER71, these cell populations contributed to alternative mesodermal lineages, including the cardiac lineage. To extend these analyses, we used an inducible embryonic stem/embryoid body system and observed that ER71 overexpression repressed cardiogenesis. Together, these studies identify ER71 as a critical regulator of mesodermal fate decisions that acts to specify the hematopoietic and endothelial lineages at the expense of cardiac lineages. This enhances our understanding of the mechanisms that govern mesodermal fate decisions early during embryogenesis.
Rasmussen, Tara L.; Kweon, Junghun; Diekmann, Mackenzie A.; Belema-Bedada, Fikru; Song, Qingfeng; Bowlin, Kathy; Shi, Xiaozhong; Ferdous, Anwarul; Li, Tongbin; Kyba, Michael; Metzger, Joseph M.; Koyano-Nakagawa, Naoko; Garry, Daniel J.
Epidermal cells in the root of Arabidopsis seedling differentiate either as hair or non-hair cells, while in the hypocotyl they become either stomatal or elongated cells. WEREWOLF (WER) and GLABRA2 (GL2) are positive regulators of non-hair and elongated cell development. CAPRICE (CPC) is a positive regulator of hair cell development in the root. We show that WER, GL2 and CPC are expressed and active during the stages of embryogenesis when the pattern of cells in the epidermis of the root-hypocotyl axis forms. GL2 is first expressed in the future epidermis in the heart stage embryo and its expression is progressively restricted to those cells that will acquire a non-hair identity in the transition between torpedo and mature stage. The expression of GL2 at the heart stage requires WER function. WER and CPC are transiently expressed throughout the root epidermal layer in the torpedo stage embryo when the cell-specific pattern of GL2 expression is being established in the epidermis. We also show that WER positively regulates CPC transcription and GL2 negatively regulates WER transcription in the mature embryo. We propose that the restriction of GL2 to the future non-hair cells in the root epidermis can be correlated with the activities of WER and CPC during torpedo stage. In the embryonic hypocotyl we show that WER controls GL2 expression. We also provide evidence indicating that CPC may also regulate GL2 expression in the hypocotyl. PMID:12756173
Costa, Silvia; Dolan, Liam
The hepatic ?-tocopherol transfer protein (TTP) is required for optimal ?-tocopherol bioavailability in humans; mutations in the human TTPA gene result in the heritable disorder ataxia with vitamin E deficiency (AVED, OMIM #277460). TTP is also expressed in mammalian uterine and placental cells and in the human embryonic yolk-sac, underscoring TTP’s significance during fetal development. TTP and vitamin E are essential for productive pregnancy in rodents, but their precise physiological role in embryogenesis is unknown. We hypothesize that TTP is required to regulate delivery of ?-tocopherol to critical target sites in the developing embryo. We tested to find if TTP is essential for proper vertebrate development, utilizing the zebrafish as a non-placental model. We verify that TTP is expressed in the adult zebrafish and its amino acid sequence is homologous to the human ortholog. We show that embryonic transcription of TTP mRNA increases >7-fold during the first 24 hours following fertilization. In situ hybridization demonstrates that Ttpa transcripts are localized in the developing brain, eyes and tail bud at 1-day post fertilization. Inhibiting TTP expression using oligonucleotide morpholinos results in severe malformations of the head and eyes in nearly all morpholino-injected embryos (88% compared with 5.6% in those injected with control morpholinos or 1.7% in non-injected embryos). We conclude that TTP is essential for early development of the vertebrate central nervous system.
Miller, Galen W.; Ulatowski, Lynn; Labut, Edwin M.; Lebold, Katie M.; Manor, Danny; Atkinson, Jeffrey; Barton, Carrie L.; Tanguay, Robert L.; Traber, Maret G.
A widely conserved aspect of embryogenesis is the ability to synchronize nuclear divisions post-fertilization. How is synchronization achieved? Given a typical protein diffusion constant of 10 ?m^2sec, and an embryo length of 1mm, it would take diffusion many hours to propagate a signal across the embryo. Therefore, synchrony cannot be attained by diffusion alone. We hypothesize that known autocatalytic reactions of cell-cycle components make the embryo an ``active medium'' in which waves propagate much faster than diffusion, enforcing synchrony. We report on robust spatial synchronization of components of the core cell cycle circuit based on a mathematical model previously determined by in vitro experiments. In vivo, synchronized divisions are preceded by a rapid calcium wave that sweeps across the embryo. Experimental evidence supports the hypothesis that increases in transient calcium levels lead to derepression of a negative feedback loop, allowing cell divisions to start. Preliminary results indicate a novel relationship between the speed of the initial calcium wave and the ability to achieve synchronous cell divisions.
McIsaac, R. Scott; Huang, K. C.; Sengupta, Anirvan; Wingreen, Ned
Increased free radical production, due to ischemia and reperfusion, has been postulated as a cause of cocaine's (COC) developmental toxicity. Salicylate reacts with hydroxyl free radicals (*OH) to form stable, quantifiable reaction products, which can be measured with high-pressure liquid chromatography (HPLC). To determine if chicken embryos' brains and hearts were exposed to increased *OH concentrations after injection of COC, an injection of a nontoxic dose of sodium salicylate (NaSAL, 100 mg/kg egg, or 5 mg/egg), followed by 5 injections of COC (13.5 mg/kg or 0.675 mg/egg, every 1.5 h), was administered to eggs containing embryos on the 12th day of embryogenesis (E12). In addition to finding increased *OH concentrations in E12 embryonic hearts and brains, we observed that the developmental toxicity of COC, manifest as vascular disruption (hemorrhage) and lethality, was enhanced by NaSAL injection. These results confirm and extend results of similar experiments performed upon older embryos (E18), and indicate that increased &z.rad;OH concentration in embryonic tissues after COC exposure and toxic interactions of COC and NaSAL can also occur at an earlier stage of development. The results are discussed in light of possible exposure of human fetuses to both COC and salicylates, since COC-abusing pregnant women can be misdiagnosed with pre-eclampsia and aspirin is used to treat this syndrome. PMID:11163537
Venturini, L; Sparber, S B
In this essay, I attempt to provide answers to the following four queries concerning the metaphysics of early human embryogenesis. (1) Following its first cellular fission, is it coherent to claim that one and only one of two "blastomeric" twins of a human zygote is identical with that zygote? (2) Following the fusion of two human pre-embryos, is it coherent to claim that one and only one pre-fusion pre-embryo is identical with that postfusion pre-embryo? (3) Does a live human being come into existence only when its brain comes into existence? (4) At implantation, does a pre-embryo become a mere part of its mother? I argue that either if things have quidditative properties or if criterialism is false, then queries (1) and (2) can be answered in the affirmative; that in light of recent developments in theories of human death and in light of a more "functional" theory of brains, query (3) can be answered in the negative; and that plausible mereological principles require a negative answer to query (4). PMID:18480498
Howsepian, A A
The hepatic ?-tocopherol transfer protein (TTP) is required for optimal ?-tocopherol bioavailability in humans; mutations in the human TTPA gene result in the heritable disorder ataxia with vitamin E deficiency (AVED, OMIM #277460). TTP is also expressed in mammalian uterine and placental cells and in the human embryonic yolk-sac, underscoring TTP's significance during fetal development. TTP and vitamin E are essential for productive pregnancy in rodents, but their precise physiological role in embryogenesis is unknown. We hypothesize that TTP is required to regulate delivery of ?-tocopherol to critical target sites in the developing embryo. We tested to find if TTP is essential for proper vertebrate development, utilizing the zebrafish as a non-placental model. We verify that TTP is expressed in the adult zebrafish and its amino acid sequence is homologous to the human ortholog. We show that embryonic transcription of TTP mRNA increases >7-fold during the first 24 hours following fertilization. In situ hybridization demonstrates that Ttpa transcripts are localized in the developing brain, eyes and tail bud at 1-day post fertilization. Inhibiting TTP expression using oligonucleotide morpholinos results in severe malformations of the head and eyes in nearly all morpholino-injected embryos (88% compared with 5.6% in those injected with control morpholinos or 1.7% in non-injected embryos). We conclude that TTP is essential for early development of the vertebrate central nervous system. PMID:23077608
Miller, Galen W; Ulatowski, Lynn; Labut, Edwin M; Lebold, Katie M; Manor, Danny; Atkinson, Jeffrey; Barton, Carrie L; Tanguay, Robert L; Traber, Maret G
To gain insights into coordinated lineage-specification and morphogenetic processes during early embryogenesis, here we report a systematic identification of transcriptional programs mediated by a key developmental regulator—Brachyury. High-resolution chromosomal localization mapping of Brachyury by ChIP sequencing and ChIP-exonuclease revealed distinct sequence signatures enriched in Brachyury-bound enhancers. A combination of genome-wide in vitro and in vivo perturbation analysis and cross-species evolutionary comparison unveiled a detailed Brachyury-dependent gene-regulatory network that directly links the function of Brachyury to diverse developmental pathways and cellular housekeeping programs. We also show that Brachyury functions primarily as a transcriptional activator genome-wide and that an unexpected gene-regulatory feedback loop consisting of Brachyury, Foxa2, and Sox17 directs proper stem-cell lineage commitment during streak formation. Target gene and mRNA-sequencing correlation analysis of the Tc mouse model supports a crucial role of Brachyury in up-regulating multiple key hematopoietic and muscle-fate regulators. Our results thus chart a comprehensive map of the Brachyury-mediated gene-regulatory network and how it influences in vivo developmental homeostasis and coordination.
Lolas, Macarena; Valenzuela, Pablo D. T.; Tjian, Robert; Liu, Zhe
Gametogenesis fertilization and early embryogenesis are crucial periods for normal development afterwards In past three decades many experiments have been conducted in space and in simulated weightlessness induced by clinostats to elucidate the issue Different animal species including Drosophila wasp shrimp fish amphibian mouse rats etc have been used for the study Oogenesis and spermatogenesis are affected by microgravity in different ways Some researches found that microgravity condition perturbed the process of oogenesis in many species A significant increased frequency of chromosomal non-disjunction was found in Drosophila females resulting the loss of chromosomes during meiosis and inhibition of cell division Studies on wasp showed a decreased hatchability and accumulation of unhatched eggs when the insects were exposed to spaceflight at different stages of oogenesis For experiments conducted on vertebrate animal models the results are somehow different however Microgravity has no significant effect for fish Medaka etc amphibian South African clawed toad Xenopus laevis or mammals mouse Spermatogenesis on the other hand is more significantly affected by microgravity condition Some researches indicated sperm are sensitive to changes in gravitational force and this sensitivity affects the ability of sperm to fertilize eggs Sperm swim with higher velocity in microgravity which is coupled with altered protein phosphorylation level in sperm under microgravity condition Microgravity also induced activation of the
Developmental biology studies, using gastrula-arrested cysts of the brine shrimp Artemia franciscana, were conducted during two flights of the space shuttle Atlantis (missions STS-37 and STS-43) in 1991. Dehydrated cysts were activated, on orbit, by addition of salt water to the cysts, and then development was terminated by the addition of fixative. Development took place in 5 ml syringes, connected by tubing to activation syringes, containing salt water, and termination syringes, containing fixative. Comparison of space results with simultaneous ground control experiments showed that equivalent percentages of naupliar larvae hatched in the syringes (40%). Thus, reactivation of development, completion of embryogenesis, emergence and hatching took place, during spaceflight, without recognizable alteration in numbers of larvae produced. Post-hatching larval development was studied in experiments where development was terminated, by introduction of fixative, 2 days, 4 days, and 8 days after reinitiation of development. During spaceflight, successive larval instars or stages, interrupted by molts, occurred, generating brine shrimp at appropriate larval instars. Naupliar larvae possessed the single naupliar eye, and development of the lateral pair of adult eyes also took place in space. Transmission electron microscopy revealed extensive differentiation, including skeletal muscle and gut endoderm, as well as the eye tissues. These studies demonstrate the potential value of Artemia for developmental biology studies during spa ceflight, and show that extensive degrees of development can take place in this microgravity environment.
Spooner, B. S.; Debell, L.; Armbrust, L.; Guikema, J. A.; Metcalf, J.; Paulsen, A.
Developmental biology studies, using gastrula-arrested cysts of the brine shrimp Artemia franciscana, were conducted during two flights of the space shuttle Atlantis (missions STS-37 and STS-43) in 1991. Dehydrated cysts were activated, on orbit, by addition of salt water to the cysts, and then development was terminated by the addition of fixative. Development took place in 5 ml syringes, connected by tubing to activation syringes, containing salt water, and termination syringes, containing fixative. Comparison of space results with simultaneous ground control experiments showed that equivalent percentages of naupliar larvae hatched in the syringes (40%). Thus, reactivation of development, completion of embryogenesis, emergence and hatching took place, during spaceflight, without recognizable alteration in numbers of larvae produced. Post-hatching larval development was studied in experiments where development was terminated, by intrduction of fixative, 2 days, 4 days, and 8 days after reinitiation of development. During spaceflight, successive larval instars or stages, interrupted by molts, occurred, generating brine shrimp at appropriate larval instars. Naupliar larvae possessed the single naupliar eye, and development of the lateral pair of adult eyes also took place in space. Transmission electron microscopy revealed extensive differentiation, including skeletal muscle and gut endoderm, as well as the eye tissues. These studies demonstrate the potential value of Artemia for developmental biology studies during spaceflight, and show that extensive degress of development can take place in this microgravity environment.
Spooner, B. S.; Debell, L.; Armbrust, L.; Guikema, J. A.; Metcalf, J.; Paulsen, A.
Objective To examine the association between biological stress regulation and somatic complaints in young girls prior to the onset of clear psychopathology such as somatization disorder. Methods Salivary cortisol, heart rate variability (HRV), and negative mood were assessed in 48 12-year-old girls in response to the Trier Social Stress Test for Children (TSST-C). Parent and child report on the Children's Somatization Inventory was used to identify girls with high and low somatic complaints. Results Girls with high levels of somatic complaints had significantly higher initial levels of cortisol, which decreased over time, and showed a trend for a more limited HRV in response to the TSST-C than girls with low levels of somatic complaints. Conclusions High levels of cortisol and possibly low HRV among girls with somatic complaints may interfere with flexibility in responding to typical psychosocial stressors, which may increase vulnerability to the onset of somatic illnesses in females.
Keenan, Kate; Marsland, Anna
An induction voidmeter for detecting voids in a conductive fluid may comprise: a four arm bridge circuit having two adjustable circuit elements connected as opposite arms of said bridge circuit, an input branch, and an output branch; two induction coils, bifilarly wound together, connected as the remaining two opposing arms of said bridge circuit and positioned such that the conductive fluid passes through said coils; applying an AC excitation signal to said input branch; and detecting the output signal generated in response to said excitation signal across said output branch. The induction coils may be located outside or inside a non-magnetic pipe containing the conductive fluid.
Anderson, Thomas T. (Downers Grove, IL) [Downers Grove, IL; Roop, Conard J. (Lockport, IL) [Lockport, IL; Schmidt, Kenneth J. (Midlothian, IL) [Midlothian, IL; Brewer, John (Oak Lawn, IL) [Oak Lawn, IL
An induction voidmeter for detecting voids in a conductive fluid may comprise: a four arm bridge circuit having two adjustable circuit elements connected as opposite arms of said bridge, an input branch, and an output branch; two induction coils, bifilarly wound together, connected as the remaining two opposing arms of said bridge circuit and positioned such that the conductive fluid passes through said coils; means for applying an AC excitation signal to said input branch; and means for detecting the output signal generated in response to said excitation signal across said output branch. The induction coils may be located outside or inside a non-magnetic pipe containing the conductive fluid.
Anderson, T.T.; Roop, C.J.; Schmidt, K.J.; Brewer, J.
Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal. Retroviral and transposon mutagenesis systems in mice have been designed and deployed in somatic tissues for surveying hematopoietic and solid tumor formation. In the context of cancer, the ability to visually mark mutant cells would present tremendous advantages for identifying tumor formation, monitoring tumor growth over time, and tracking tumor infiltrations and metastases into wild-type tissues. Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes. For this purpose, we developed a system using the piggyBac (PB) transposon for somatic mutagenesis with an activated reporter and tracker, called PB-SMART. The PB-SMART mouse genetic screening system can simultaneously induce somatic mutations and mark mutated cells using bioluminescence or fluorescence. The marking of mutant cells enable analyses that are not possible with current somatic mutagenesis systems, such as tracking cell proliferation and tumor growth, detecting tumor cell infiltrations, and reporting tissue mutagenesis levels by a simple ex vivo visual readout. We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions. We further integrated a conditional transposase with the PB-SMART system, permitting tissue-specific mutagenesis with a single cross to any available Cre line. Targeting the germline, the system could also be used to conduct F1 screens. With these features, PB-SMART provides an integrated platform for individual investigators to harness the power of somatic mutagenesis and phenotypic screens to decipher the genetic basis of mammalian biology and disease.
Bosenberg, Marcus W.; Xu, Tian
The paper discusses a mechanism for short-long duplications of segments of somatic chromatids in cancer cells. Based on the Frenkel-Kontorova model, the mechanism accounts for the high incidence of duplications on the long arm of almost all chromatids. PMID:10052868
Matioli, G T
Childhood exposure to trauma has been associated with increased rates of somatic symptoms (SS), which may contribute to diminished daily functioning. One hundred and sixty-one children residing at a residential treatment home who had experienced neglect and/or abuse were administered the Trauma Symptom Checklist for Children (TSCC), the…
Kugler, Brittany B.; Bloom, Marlene; Kaercher, Lauren B.; Truax, Tatyana V.; Storch, Eric A.
Discusses the somatic mind, a permeable materiality in which mind and body resolve into a single entity which is (re)formed by the constantly shifting boundaries of discursive and corporeal intertextualities. Addresses its importance in composition studies. Critiques the poststructuralist disregard of corporeality. (CR)
Fleckenstein, Kristie S.
The substantia nigra (SN) pars compacta is arranged somatotopically. Symptoms of idiopathic Parkinson's disease (IPD) are caused by a lesion in this nucleus, which spreads in a stereotyped spatio-temporal pattern during the course of the disease. We investigated the order of somatic symptom progression in a group of 30 patients with IPD to determine if progression of symptoms was consistent
Jon M. Dickson; Richard A. Grünewald
Immunoglobulin (Ig) diversification by somatic hypermutation in germinal center B cells is instrumental for maturation of the humoral immune response, but also bears the risk of excessive or aberrant genetic changes. Thus, introduction of DNA damage by activation-induced cytidine deaminase as well as DNA repair by multiple pathways need to be tightly regulated during the germinal center response to prevent lymphomagenesis. In the present study, we show that DNA damage checkpoint signaling via checkpoint kinase 1 (Chk1) negatively regulates somatic hypermutation. Chk1 inhibition in human B cell lymphoma lines as well as inactivation of Chk1 alleles by gene targeting in DT40 B cells leads to increased somatic hypermutation. This is apparently due to changes in DNA repair pathways regulated by Chk1, such as a decreased homologous recombination efficiency that also leads to decreased Ig gene conversion in DT40. Our data show that Chk1 signaling plays a crucial role in regulation of Ig diversification and sheds unexpected light on potential origins of aberrant somatic hypermutation in B cell lymphomagenesis. PMID:24423870
Frankenberger, Samantha; Davari, Kathrin; Fischer-Burkart, Sabine; Böttcher, Katrin; Tomi, Nils-Sebastian; Zimber-Strobl, Ursula; Jungnickel, Berit
Chart reviews and telephone interviews with 72 sexual abuse victims found that 48 of the children had symptoms similar to the "rape trauma" syndrome. Two-thirds of victims commonly had somatic complaints (such as abdominal pain) and emotional/behavioral problems (runaway behavior, suicide attempts). (DB)
Rimza, Mary Ellen; And Others
Briefly reviews number of theories which address role of psychological factors in etiology of somatic disorders. Focuses on psychological treatment approaches that have been used to alleviate or reduce symptomatic behaviors associated with eating disorders, elimination disorders, and headaches in children. Discusses role of school psychologists in…
Siegel, Lawrence J.
A psychosomatic syndrome is defined as a syndrome in which psychological processes play a substantial role in the etiology of the illness in some of the patients. The main conclusions on the extent of the biological and psychosocial contributions to several psychosomatic syndromes are presented and the relationship of these syndromes to somatization and somatoform disorders is discussed. The syndromes
The existence of human endometrial somatic stem cells was proposed in the mid-20th century for the first time. This hypothesis became stronger and was revised by two authors between 1978 and 1989. Nevertheless, it was not until 2004 that scientific evidence was first published. As we describe here, the great regenerative capability of the human endometrium has been finally questioned in the last 8 years, and this period can be considered the most productive in endometrial stem cell biology given the new scientific information recapitulated to date. We provide a detailed summary based on the actual scientific knowledge obtained about (1) the existence of somatic stem cells in murine (detected with label-retaining cell methods) and human (cells isolated by different methods) endometria, (2) the involvement of bone marrow as a putative extrauterine source of endometrial somatic stem cells, (3) the implication and biological pathways of these cells in several pathologies like endometriosis and endometrial cancer, and (4) the future of endometrial somatic stem cells in regenerative medicine to provide new strategies in autologous transplant and bioengineering. PMID:23329639
Cervelló, Irene; Mas, Aymara; Gil-Sanchis, Claudia; Simón, Carlos
The techniques described permit the controlled production of large numbers of prolifer- ating somatic cell hybrids in a relatively short period of time . Sendai virus is used to pro- mote cell hybridization . ß-propriolactone is employed as the inactivating agent of Sendai virus since it produces complete loss of viral infectivity while preserving viral fusion capac- ity. Cells are
ROBERT J. KLEBE; T Chen; F H Ruddle
In contrast to many other genes containing a CpG island, the testis-specific H2B (TH2B) histone gene exhibits tissue-specific methylation patterns in correlation with gene activity. Characterization of the methylation patterns within a 20-kb segment containing the TH2A and TH2B genes in comparison with that in a somatic histone cluster revealed that: (i) the germ cell-specific unmethylated domain of the TH2A and TH2B genes is defined as a small region surrounding the CpG islands of the TH2A and TH2B genes and (ii) somatic histone genes are unmethylated in both liver and germ cells, like other genes containing CpG islands, whereas flanking sequences are methylated. Transfection of in vitro-methylated TH2B, somatic H2B, and mouse metallothionein I constructs into F9 embryonal carcinoma cells revealed that the CpG islands of the TH2A and TH2B genes were demethylated like those of the somatic H2A and H2B genes and the metallothionein I gene. The demethylation of those CpG islands became significantly inefficient at a high number of integrated copies and a high density of methylated CpG dinucleotides. In contrast, three sites in the somatic histone cluster, of which two sites are located in the long terminal repeat of an endogenous retrovirus-like sequence, were efficiently demethylated even at a high copy number and a high density of methylated CpG dinucleotides. These results suggest two possible mechanisms for demethylation in F9 cells and methylation of CpG islands of the TH2A and TH2B genes at the postblastula stage during embryogenesis. Images
Choi, Y C; Chae, C B
Angiosperm and gymnosperm plants evolved from a common ancestor about 300 million years ago. Apart from morphological and structural differences in embryogenesis and seed origin, a set of embryogenesis-regulating genes and the molecular mechanisms involved in embryo development seem to have been conserved alike in both taxa. Few studies have covered molecular aspects of embryogenesis in the Brazilian pine, the only economically important native conifer in Brazil. Thus eight embryogenesis-regulating genes, viz., ARGONAUTE 1, CUP-SHAPED COTYLEDON 1, WUSCHEL-related WOX, S-LOCUS LECTIN PROTEIN KINASE, SCARECROW-like, VICILIN 7S, LEAFY COTYLEDON 1, and REVERSIBLE GLYCOSYLATED POLYPEPTIDE 1, were analyzed through semi-quantitative RT-PCR during embryo development and germination. All the eight were found to be differentially expressed in the various developmental stages of zygotic embryos, seeds and seedling tissues. To our knowledge, this is the first report on embryogenesis-regulating gene expression in members of the Araucariaceae family, as well as in plants with recalcitrant seeds. PMID:22481892
Schlögl, Paulo Sérgio; Dos Santos, André Luis Wendt; Vieira, Leila do Nascimento; Floh, Eny Iochevet Segal; Guerra, Miguel Pedro
Angiosperm and gymnosperm plants evolved from a common ancestor about 300 million years ago. Apart from morphological and structural differences in embryogenesis and seed origin, a set of embryogenesis-regulating genes and the molecular mechanisms involved in embryo development seem to have been conserved alike in both taxa. Few studies have covered molecular aspects of embryogenesis in the Brazilian pine, the only economically important native conifer in Brazil. Thus eight embryogenesis-regulating genes, viz., ARGONAUTE 1, CUP-SHAPED COTYLEDON 1, WUSCHEL-related WOX, S-LOCUS LECTIN PROTEIN KINASE, SCARECROW-like, VICILIN 7S, LEAFY COTYLEDON 1, and REVERSIBLE GLYCOSYLATED POLYPEPTIDE 1, were analyzed through semi-quantitative RT-PCR during embryo development and germination. All the eight were found to be differentially expressed in the various developmental stages of zygotic embryos, seeds and seedling tissues. To our knowledge, this is the first report on embryogenesis-regulating gene expression in members of the Araucariaceae family, as well as in plants with recalcitrant seeds.
Schlogl, Paulo Sergio; dos Santos, Andre Luis Wendt; Vieira, Leila do Nascimento; Floh, Eny Iochevet Segal; Guerra, Miguel Pedro
The bdelloid rotifer Adineta ricciae is an asexual microinvertebrate that can survive desiccation by entering an ametabolic state known as anhydrobiosis. Two late embryogenesis abundant (LEA) proteins, ArLEA1A and ArLEA1B, have been hypothesized to contribute to desiccation tolerance in these organisms, since in vitro assays suggest that ArLEA1A and ArLEA1B stabilize desiccation-sensitive proteins and membranes, respectively. To examine their functions in vivo, it is important to analyse the cellular distribution of the bdelloid LEA proteins. Bioinformatics predicted their translocation into the endoplasmic reticulum (ER) via an N-terminal ER translocation signal and persistence in the same compartment via a variant C-terminal retention signal sequence ATEL. We assessed the localization of LEA proteins in bdelloids and in a mammalian cell model. The function of the N-terminal sequence of ArLEA1A and ArLEA1B in mediating ER translocation was verified, but our data showed that, unlike classical ER-retention signals, ATEL allows progression from the ER to the Golgi and limited secretion of the proteins into the extracellular medium. These results suggest that the N-terminal ER translocation signal and C-terminal ATEL sequence act together to regulate the distribution of rotifer LEA proteins within intracellular vesicular compartments, as well as the extracellular space. We speculate that this mechanism allows a small number of LEA proteins to offer protection to a large number of desiccation-sensitive molecules and structures both inside and outside cells in the bdelloid rotifer. PMID:22837450
Tripathi, Rashmi; Boschetti, Chiara; McGee, Brian; Tunnacliffe, Alan
The emergence of avian satellite cells during development has been studied using markers that distinguish adult from fetal cells. Previous studies by us have shown that myogenic cultures from fetal (Embryonic Day 10) and adult (12–16 weeks) chicken pectoralis muscle (PM) each regulate expression of the embryonic isoform of fast myosin heavy chain (MHC) differently. In fetal cultures, embryonic MHC is coexpressed with a ventricular MHC in both myocytes (differentiated myoblasts) and myotubes. In contrast, myocytes and newly formed myotubes in adult cultures express ventricular but not embryonic MHC. In the current study, the appearance of myocytes and myotubes which express ventricular but not embryonic MHC was used to determine when adult myoblasts first emerge during avian development. By examining patterns of MHC expression in mass and clonal cultures prepared from embryonic and posthatch chicken skeletal muscle using double-label immunofluorescence with isoform-specific monoclonal antibodies, we show that a significant number of myocytes and myotubes which stain for ventricular but not embryonic MHC are first seen in cultures derived from PM during fetal development (Embryonic Day 18) and comprise the majority, if not all, of the myoblasts present at hatching and beyond. These results suggest that adult type myoblasts become dominant in late embryogenesis. We also show that satellite cell cultures derived from adult slow muscle give results similar to those of cultures derived from adult fast muscle. Cultures derived from Embryonic Day 10 hindlimb form myocytes and myotubes that coexpress ventricular and embryonic MHCs in a manner similar to cells of the Embryonic Day 10 PM. Thus, adult and fetal expression patterns of ventricular and embryonic MHCs are correlated with developmental age but not muscle fiber type.
Hartley, Rebecca S.; Bandman, Everett
Endodermal organogenesis requires a precise orchestration of cell fate specification and cell movements, collectively coordinating organ size and shape. In Caenorhabditis elegans, uncoordinated-53 (unc-53) encodes a neural guidance molecule that directs axonal growth. One of the vertebrate homologs of unc-53 is neuron navigator 3 (Nav3). Here, we identified a novel vertebrate neuron navigator 3 isoform in zebrafish, nav3a, and we provide genetic evidence in loss- and gain-of-function experiments showing its functional role in endodermal organogenesis during zebrafish embryogenesis. In zebrafish embryos, nav3a expression was initiated at 22 hpf in the gut endoderm and at 40 hpf expanded to the newly formed liver bud. Endodermal nav3a expression was controlled by Wnt2bb signaling and was independent of FGF and BMP signaling. Morpholino-mediated knockdown of nav3a resulted in a significantly reduced liver size, and impaired development of pancreas and swim bladder. In vivo time-lapse imaging of liver development in nav3a morphants revealed a failure of hepatoblast movement out from the gut endoderm during the liver budding stage, with hepatoblasts being retained in the intestinal endoderm. In hepatocytes in vitro, nav3a acts as a positive modulator of actin assembly in lamellipodia and filipodia extensions, allowing cellular movement. Knockdown of nav3a in vitro impeded hepatocyte movement. Endodermal-specific overexpression of nav3a in vivo resulted in additional ectopic endodermal budding beyond the normal liver and pancreatic budding sites. We conclude that nav3a is required for directing endodermal organogenesis involving coordination of endodermal cell behavior. PMID:21471154
Klein, Christian; Mikutta, Janine; Krueger, Janna; Scholz, Katja; Brinkmann, Joep; Liu, Dong; Veerkamp, Justus; Siegel, Doreen; Abdelilah-Seyfried, Salim; le Noble, Ferdinand
Prevalence and structure of comorbidity a somatic pathology in military men with neurotic disorders was studied. It was established that 40,4% of surveyed noted concomitant somatic pathology, the structure of which was dominated by gastro-intestinal tract (26,8%), and pathology of the cardiovascular system (21,6%). It is shown that concomitant somatic pathology provided aggravating effect on clinic neurotic disorders in serviceman, making it difficult to diagnose mental disorders. The greatest risk concomitant a somatic pathology was marked in patients with depressive and somatoform disorders. Indicates the need for specialized standards of care for persons with comorbid mental and somatic disorders. PMID:22712246
Kurasov, E S; Marchenko, A A; Krasnov, A A; Golovach, I G; Kozlova, S N
The central role performed by billions of vital central nervous system (CNS) lipids “lipidomics” in medical physiology is usually overlooked. A metabolic deficiency embracing these vital lipids can form the aetiology for a variety of diseases. CNS lipids regulate embryogenesis, cell induction, mental balance by preventing autism spectrum disorders, depression, burn-out syndromes like posttraumatic stress disease PTSD, by guarding normal immunity, treating sterile inflammatory diatheses with a titanium containing lymphopoietic CNS lipid component. The propaganda driving for unphysiological fat-free diets is dangerous and can cause serious health problems for a whole generation. This article presents a broad list of various mental and motor bodily functions of which the healthy function depends on these vital CNS lipids. A rigorous fat-free diet can provoke these metabolic lipid deficiencies but they can fortunately be compensated by dietary supplementation, but not by pharmacologic treatment.
Tallberg, Thomas; Dabek, Jan; Hallamaa, Raija; Atroshi, Faik
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. PMID:23250407
Álvarez, José M; Cortizo, Millán; Ordás, Ricardo J
Investigation of the relative biological effectiveness of energetic protons for the induction of somatic effects in a mammal (mice) following whole body irradiation. The proton energy used approximates the mean energy for proton spectra accompanying solar events. The effects on longevity and the incidence of major neoplastic diseases are summarized. The results obtained suggest that medium energy proton irradiation is no more effective, and on the whole, probably less effective, than conventional X radiation for the induction of late radiation effects in the mouse.
Darden, E. B., Jr.; Clapp, N. K.; Bender, R. S.; Jernigan, M. C.; Upton, A. C.
Objective. To examine the effects of intraarticular induction of interleukin-1 (IL-1) expression in adult mice. Methods. We used somatic mosaic analysis in a novel transgenic mouse with an inducible IL-1 tran- scription unit. Transgene activation was induced by Cre recombinase in the temporomandibular joints (TMJs) of adult transgenic mice (conditional knockin model). The effects of intraarticular IL-1 induction were sub-
Yu-Ching Lai; Solomon S. Shaftel; Jen-nie H. Miller; Ross H. Tallents; Yoon Chang; Carl A. Pinkert; John A. Olschowka; Ian M. Dickerson; J. Edward Puzas; M. Kerry O'Banion; Stephanos Kyrkanides
We studied changes in the activities of serin, thiol, and aspartyl proteinases and their protein inhibitors during embryogenesis of the silkworm. The dynamics of the activities of the protein inhibitors and specific proteinases were interrelated, thus providing for the coordination and fine regulation of the functioning of the proteolytic complex of enzymes during embryogenesis. Possible functions of peptidohydrolases and their
D. V. Yarygin; S. M. Klunova; Yu. B. Filippovich
Summary Lamin A is an inner nuclear membrane protein that maintains nuclear structure integrity, is involved in transcription, DNA damage response and genomic stability, and also links to cell differentiation, senescence, premature aging and associated diseases. Induced pluripotent stem (iPS) cells have been successfully generated from various types of cells and used to model human diseases. It remains unclear whether levels of lamin A influence reprogramming of somatic cells to pluripotent states during iPS induction. Consistently, lamin A is expressed more in differentiated than in relatively undifferentiated somatic cells, and increases in expression levels with age. Somatic cells with various expression levels of lamin A differ in their dynamics and efficiency during iPS cell induction. Cells with higher levels of lamin A show slower reprogramming and decreased efficiency to iPS cells. Furthermore, depletion of lamin A by transient shRNA accelerates iPS cell induction from fibroblasts. Reduced levels of lamin A are associated with increased expression of pluripotent genes Oct4 and Nanog, and telomerase genes Tert and Terc. On the contrary, overexpression of lamin A retards somatic cell reprogramming to iPS-like colony formation. Our data suggest that levels of lamin A influence reprogramming of somatic cells to pluripotent stem cells and that artificial silencing of lamin A facilitates iPS cell induction. These findings may have implications in enhancing rejuvenation of senescent or older cells by iPS technology and manipulating lamin A levels.
Zuo, Bingfeng; Yang, Jiao; Wang, Fang; Wang, Lei; Yin, Yu; Dan, Jiameng; Liu, Na; Liu, Lin
Lamin A is an inner nuclear membrane protein that maintains nuclear structure integrity, is involved in transcription, DNA damage response and genomic stability, and also links to cell differentiation, senescence, premature aging and associated diseases. Induced pluripotent stem (iPS) cells have been successfully generated from various types of cells and used to model human diseases. It remains unclear whether levels of lamin A influence reprogramming of somatic cells to pluripotent states during iPS induction. Consistently, lamin A is expressed more in differentiated than in relatively undifferentiated somatic cells, and increases in expression levels with age. Somatic cells with various expression levels of lamin A differ in their dynamics and efficiency during iPS cell induction. Cells with higher levels of lamin A show slower reprogramming and decreased efficiency to iPS cells. Furthermore, depletion of lamin A by transient shRNA accelerates iPS cell induction from fibroblasts. Reduced levels of lamin A are associated with increased expression of pluripotent genes Oct4 and Nanog, and telomerase genes Tert and Terc. On the contrary, overexpression of lamin A retards somatic cell reprogramming to iPS-like colony formation. Our data suggest that levels of lamin A influence reprogramming of somatic cells to pluripotent stem cells and that artificial silencing of lamin A facilitates iPS cell induction. These findings may have implications in enhancing rejuvenation of senescent or older cells by iPS technology and manipulating lamin A levels. PMID:23213392
Zuo, Bingfeng; Yang, Jiao; Wang, Fang; Wang, Lei; Yin, Yu; Dan, Jiameng; Liu, Na; Liu, Lin
Embryogenic cultures of Daucus carota treated with 1 millimolar alpha-difluoromethylarginine, a specific inhibitor of arginine decarboxylase, exhibited nearly a 50 percent reduction in embryo formation compared with controls. Putrescine and spermidine concentrations in the treated cells were greatly reduced. Addition of putrescine, spermidine, or spermine to the culture medium restored embryogenesis in the treated cultures. Embryogenesis was not significantly affected by alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase. These results suggest that polyamines have a major function in plant embryo development and that the wild carrot synthesizes polyamines through the biosynthetic pathway involving arginine decarboxylase rather than ornithine decarboxylase. PMID:17746056
Feirer, R P; Mignon, G; Litvay, J D
We present some results of comparison studying of Brassica embryo development and the ?-cyclin genes expression under slow horizontal clinorotation and in the laboratory control. Some backlog of the ?1-cyclin genes expression at early stages of embryogenesis under clinorotation was revealed in comparison with the laboratory control. The similar level of the ?3-cyclin expression at all stages of embryo formation (from one to nine days) in both variants is shown. Some delays in the rate of Brassica rapa embryo development under clinorotation in comparison with the laboratory control can be a result of decrease of a level and some backlog of the ?1-cyclin expression at early stages of embryogenesis.
Artemenko, O. A.; Popova, A. F.
The advance in technological tools for massively parallel, high-throughput sequencing of DNA has enabled the comprehensive characterization of somatic mutations in large number of tumor samples. Here, we review recent cancer genomic studies that have assembled emerging views of the landscapes of somatic mutations through deep sequencing analyses of the coding exomes and whole genomes in various cancer types. We discuss the comparative genomics of different cancers, including mutation rates, spectrums, and roles of environmental insults that influence these processes. We highlight the developing statistical approaches used to identify significantly mutated genes, and discuss the emerging biological and clinical insights from such analyses as well as the challenges ahead translating these genomic data into clinical impacts.
Watson, Ian R.; Takahashi, Koichi; Futreal, P. Andrew; Chin, Lynda
The technology underlying genetic toxicology has undergone enormous change during the last 25 years. Technology has permitted studies of somatic mutations in humans to achieve scientific respectability. However, the author is concerned that in the zeal to develop assays for detecting mutations in humans, it was assumed that they would be useful in public health. Now that assumption must be tested so the question is no longer can we measure mutations? but why? The author discusses the challenge for in vivo somatic mutations, at least those measured by most genetic toxicologists, viz. mutations in reporter genes. These genes are irrelevant in themselves but ideal for assessing the mutation process itself. How well do surrogate mutations in surrogate tissue mimic disease gene mutations in target tissues? 28 refs.
Albertini, R.J. [Univ. of Vermont, Burlington, VT (United States)
A functional cloning approach using the oxidant-sensitive yeast mutant, Deltayap1, was employed to identify plant genes involved in tolerance of oxidative stress. In this screen, we identified an Arabidopsis late embryogenesis-abundant (LEA)-like protein, AtLEA5, which increased the tolerance of Deltayap1 cells to the oxidants H(2)O(2), diamide, menadione and tert-butyl hydroperoxide. Unlike canonical LEAs, AtLEA5 is constitutively expressed in roots and reproductive organs but not in seeds. In leaves of short-day grown plants, AtLEA5 transcripts exhibited a diurnal pattern of regulation, where transcripts were repressed in the light and abundant in the dark. Expression of AtLEA5 in leaves was induced by oxidants, ABA and dehydration. Use of abi1-1 (ABA-insensitive) and aba1-1 (ABA-deficient) Arabidopsis mutants indicated that drought induction of AtLEA5 required ABA synthesis but was independent of the ABI1 gene product. Abscisic acid and H(2)O(2) induction of AtLEA5 was also independent of the OXI1 protein kinase. Constitutive overexpression of AtLEA5 resulted in increased root growth and shoot biomass, both in optimal conditions and under H(2)O(2) stress. However, in comparison with wild type, photosynthesis in overexpressing plants was more susceptible to drought. These features suggest that AtLEA5 has a unique function among LEA proteins in that it plays a specific role in protection against oxidative stress involving decreased photosynthesis. This protein functions as part of a complex network of defences that contribute to robustness of plants under stress by minimizing the negative effects of oxidation. PMID:17092320
Mowla, Shaheen B; Cuypers, Ann; Driscoll, Simon P; Kiddle, Guy; Thomson, Jennifer; Foyer, Christine H; Theodoulou, Frederica L
Cardiovascular autonomic neuropathy (CAN) is one of the most clinically significant complications of diabetes mellitus. Even\\u000a though many ethological factors have been attributed for the pathogenesis of this disease no attempts were made to correlate\\u000a DNA damage as a causative factor. Hence the present study was undertaken to asses the extent of somatic DNA damages by cytokinesis-block\\u000a micronuclei assay (CBMN).
A. Supriya SimonD; D. Dinesh Roy; V. Jayapal; T. Vijayakumar
Even in an established illness, somatic complains can hide other emotional inquiries. The therapist, always with a kind attitude, can ask more about patient's sexual life. This can be use of having a better idea of patient's life and problems. Talking about dreams can also be useful: it gives new and surprising elements about patient's personality and helps to progress on healing's way. PMID:24734362
Here we report whole-exome sequencing of individuals with various myeloid malignancies and identify recurrent somatic mutations in SETBP1, consistent with a recent report on atypical chronic myeloid leukemia (aCML). Closely positioned somatic SETBP1 mutations encoding changes in Asp868, Ser869, Gly870, Ile871 and Asp880, which match germline mutations in Schinzel-Giedion syndrome (SGS), were detected in 17% of secondary acute myeloid leukemias (sAML) and 15% of chronic myelomonocytic leukemia (CMML) cases. These results from deep sequencing demonstrate a higher mutational detection rate than reported with conventional sequencing methodology. Mutant cases were associated with advanced age and monosomy 7/deletion 7q (-7/del(7q)) constituting poor prognostic factors. Analysis of serially collected samples indicated that SETBP1 mutations were acquired during leukemic evolution. Transduction with mutant Setbp1 led to the immortalization of mouse myeloid progenitors that showed enhanced proliferative capacity compared to cells transduced with wild-type Setbp1. Somatic mutations of SETBP1 seem to cause gain of function, are associated with myeloid leukemic transformation and convey poor prognosis in myelodysplastic syndromes (MDS) and CMML. PMID:23832012
Makishima, Hideki; Yoshida, Kenichi; Nguyen, Nhu; Przychodzen, Bartlomiej; Sanada, Masashi; Okuno, Yusuke; Ng, Kwok Peng; Gudmundsson, Kristbjorn O; Vishwakarma, Bandana A; Jerez, Andres; Gomez-Segui, Ines; Takahashi, Mariko; Shiraishi, Yuichi; Nagata, Yasunobu; Guinta, Kathryn; Mori, Hiraku; Sekeres, Mikkael A; Chiba, Kenichi; Tanaka, Hiroko; Muramatsu, Hideki; Sakaguchi, Hirotoshi; Paquette, Ronald L; McDevitt, Michael A; Kojima, Seiji; Saunthararajah, Yogen; Miyano, Satoru; Shih, Lee-Yung; Du, Yang; Ogawa, Seishi; Maciejewski, Jaroslaw P
Specialized ribonucleoprotein organelles collectively known as germ granules are found in the germline cytoplasm from worms to humans . In Drosophila, germ granules have been implicated in germline determination . C. elegans germ granules, known as P granules, do not appear to be required for primordial germ cell (PGC) determination , but their components are still needed for fertility [4-6]. One potential role for P granules is to maintain germline fate and totipotency. This is suggested by the loss of P granules from germ cells that transform into somatic cell types, e.g., in germlines lacking MEX-3 and GLD-1 or upon neuronal induction by CHE-1 [7, 8]. However, it has not been established whether loss of P granules is the cause or effect of cell fate transformation. To test cause and effect, we severely compromised P granules by simultaneously knocking down factors that nucleate granule formation (PGL-1 and PGL-3) and promote their perinuclear localization (GLH-1 and GLH-4)  and investigated whether this causes germ cells to lose totipotency and initiate somatic reprogramming. We found that compromising P granules causes germ cells to express neuronal and muscle markers and send out neurite-like projections, suggesting that P granules maintain totipotency and germline identity by antagonizing somatic fate. PMID:24746798
Updike, Dustin L; Knutson, Andrew Kek?pa'a; Egelhofer, Thea A; Campbell, Anne C; Strome, Susan
The discovery of induced pluripotent stem (iPS) cells provides not only new approaches for cell replacement therapy, but also new ways for drug screening. However, the undefined mechanism and relatively low efficiency of reprogramming have limited the application of iPS cells. In an attempt to further optimize the reprogramming condition, we unexpectedly observed that removing c-Myc from the Oct-4, Sox-2, Klf-4, and c-Myc (OSKM) combination greatly enhanced the generation of iPS cells. The iPS cells generated without c-Myc attained salient pluripotent characteristics and were capable of producing full-term mice through tetraploid complementation. We observed that forced expression of c-Myc induced the expression of many genes involved in cell cycle control and a hyperproliferation state of the mouse embryonic fibroblasts during the early stage of reprogramming. This enhanced proliferation of mouse embryonic fibroblasts correlated negatively to the overall reprogramming efficiency. By applying small molecule inhibitors of cell proliferation at the early stage of reprogramming, we were able to improve the efficiency of iPS cell generation mediated by OSKM. Our data demonstrated that the proliferation rate of the somatic cell plays critical roles in reprogramming. Slowing down the proliferation of the original cells might be beneficial to the induction of iPS cells. PMID:23439651
Xu, Yongyu; Wei, Xiaoyuan; Wang, Min; Zhang, Ru; Fu, Yanbin; Xing, Mingzhe; Hua, Qiuhong; Xie, Xin
There is ample epidemiological evidence showing that sunlight can cause skin cancer in the human. In experimental studies, simulated sunlight or UV lamps are used for demonstrating carcinogenesis and other biological effects. Little studies, however, have been performed using natural sunlight itself. In this work, we have examined the mutagenicity of natural sunlight in Drosophila. The Drosophila wing spot test is useful to detect somatic cell mutations. Third instar larvae in petri dishes were exposed to sunlight (ultraviolet region with < 290 nm wavelength cut off by a plastic cover) in the yard of Okayama University campus (north latitude: 34 degrees 39', east longitude: 133 degrees 55'). The sunlight was mutagenic in Drosophila larvae and produced pyrimidine dimers in their DNA. In the observed mutagenicity, there was dependence on the exposure period and UV fluence. During the two-year monitoring, the highest induction of mutant spot observed was 1.98 total spots/wing on June 25, 1998, and the lowest was 0.64 on December 29, 1998, while non-exposure spontaneous spots were 0.29 and 0.32 on these days, respectively. Thus, solar radiation was mutagenic both in summer and in winter. PMID:10709352
Negishi, T; Takinami, S; Nikaido, O; Mochizuki, M; Toyoshima, M
The discovery of induced pluripotent stem (iPS) cells provides not only new approaches for cell replacement therapy, but also new ways for drug screening. However, the undefined mechanism and relatively low efficiency of reprogramming have limited the application of iPS cells. In an attempt to further optimize the reprogramming condition, we unexpectedly observed that removing c-Myc from the Oct-4, Sox-2, Klf-4, and c-Myc (OSKM) combination greatly enhanced the generation of iPS cells. The iPS cells generated without c-Myc attained salient pluripotent characteristics and were capable of producing full-term mice through tetraploid complementation. We observed that forced expression of c-Myc induced the expression of many genes involved in cell cycle control and a hyperproliferation state of the mouse embryonic fibroblasts during the early stage of reprogramming. This enhanced proliferation of mouse embryonic fibroblasts correlated negatively to the overall reprogramming efficiency. By applying small molecule inhibitors of cell proliferation at the early stage of reprogramming, we were able to improve the efficiency of iPS cell generation mediated by OSKM. Our data demonstrated that the proliferation rate of the somatic cell plays critical roles in reprogramming. Slowing down the proliferation of the original cells might be beneficial to the induction of iPS cells.
Xu, Yongyu; Wei, Xiaoyuan; Wang, Min; Zhang, Ru; Fu, Yanbin; Xing, Mingzhe; Hua, Qiuhong; Xie, Xin
A review of the current literature suggests that comorbidity exists between depression, somatization, and suicidal ideation among behaviorally disruptive adolescents. A study was designed to determine how these clinical characteristics and other clinical indicators measured by the Millon Adolescent Clinical Inventory differentiate between male detained juvenile offenders who somatize and those that do not. A combination of analysis of variance
Brian A Glaser; Georgia B Calhoun; John V Petrocelli; Jeffrey M Bates; Lisa A Owens-Hennick
Class IIa histone deacetylases (HDACs) and myocyte enhancer factor 2 (MEF2) proteins compose a signaling module that orchestrates lineage specification during embryogenesis. We show here that this module also regulates the generation of mouse induced pluripotent stem cells by defined transcription factors. Class IIa HDACs and MEF2 proteins rise steadily during fibroblast reprogramming to induced pluripotent stem cells. MEF2 proteins tend to block the process by inducing the expression of Tgf? cytokines, which impairs the necessary phase of mesenchymal-to-epithelial transition (MET). Conversely, class IIa HDACs endeavor to suppress the activity of MEF2 proteins, thus enhancing the MET and colony formation efficiency. Our work highlights an unexpected role for a developmental axis in somatic cell reprogramming and provides new insight into how the MET is regulated in this context.
Zhuang, Qiang; Qing, Xiaobing; Ying, Yue; Wu, Haitao; Benda, Christina; Lin, Jiao; Huang, Zhijian; Liu, Longqi; Xu, Yan; Bao, Xichen; Qin, Baoming; Pei, Duanqing; Esteban, Miguel A.
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
Erhard Kranz; Horst Lor
BACKGROUND: LEA (late embryogenesis abundant) proteins have first been described about 25 years ago as accumulating late in plant seed development. They were later found in vegetative plant tissues following environmental stress and also in desiccation tolerant bacteria and invertebrates. Although they are widely assumed to play crucial roles in cellular dehydration tolerance, their physiological and biochemical functions are largely
Michaela Hundertmark; Dirk K Hincha
Summary In the giant cells of thePhaseolus coccineus embryo suspensor the number of nucleoli is not constant during early embryogenesis. Over the same period the number and the type of chromosomes involved in the nucleolar organization also vary. Moreover, the percentage of DNA and RNA puffs organized by ribosomal cistron (rc) bearing regions changes. When single chromosome regions bearingrc are
M. Frediani; L. M. C. Forino; A. M. Tagliasacchi; P. G. Cionini; M. Durante; S. Avanzi
The Drosophila Pax6 homolog twin of eyeless (toy) is so far the first zygotically expressed gene involved in eye morphogenesis in Drosophila. The study of its expression during embryogenesis is therefore informative of the initial events of eye development in Drosophila. We have analyzed how the initial expression domain of toy at cellular blastoderm is regulated. We show that the
Jorge Blanco; Walter J. Gehring
BACKGROUND: Morphogenetic events that shape the Drosophila melanogaster embryo are tightly controlled by a genetic program in which specific sets of genes are up-regulated. We used a suppressive subtractive hybridization procedure to identify a group of developmentally regulated genes during early stages of D. melanogaster embryogenesis. We studied the spatiotemporal activity of these genes in five different intervals covering 12
Alejandro Zúñiga; Christian Hödar; Patricia Hanna; Freddy Ibáñez; Pablo Moreno; Rodrigo Pulgar; Luis Pastenes; Mauricio González; Verónica Cambiazo
Summary In anther cultures ofDatura metel, pollen-embryoids could be induced on a medium consisting of honey alone. Further studies revealed that a combination of fructose and dextrose was sufficient to trigger pollen-embryogenesis. The 2 sugars, when tried individually, did not elicit any response.
S. K. Raina; R. D. Iyer
Summary In recent years, the rapid development of somatic cell genetics has made possible the transfer of alien genes over wide taxonomic\\u000a distances by somatic hybridization. In this review, the potential of somatic hybridization in the breeding of crops within\\u000a the Brassicaceae, Fabaceae, Poaceae and Solanaceae is discussed. It is evident from these studies that many hybrids, either\\u000a symmetric or asymmetric,
Sylvia Waara; Kristina Glimelius
A polyphase rotary induction machine for use as a motor or generator utilizing a single rotor assembly having two series connected sets of rotor windings, a first stator winding disposed around the first rotor winding and means for controlling the current induced in one set of the rotor windings compared to the current induced in the other set of the rotor windings. The rotor windings may be wound rotor windings or squirrel cage windings.
Owen, Whitney H. (Ogden, UT)
During the Persian Gulf war the Israeli civilian population was the target of missile attacks that could have carried poison gas warheads. During the attacks all civilians were ordered to wear gas masks and move into sealed rooms. Four telephone surveys studied the somatic reactions to this anxiety-arousing situation. The percent of subjects reporting the somatic reactions declined from 38% after the first attack to 20% 12 days later. Somatic reactions were more prevalent among females than males, and among those who completed elementary school only compared to those with an academic education. A complex pattern of relationships between expectancies about chemical warfare attacks and somatic reactions is discussed. PMID:1757246
Carmeli, A; Liberman, N; Mevorach, L
Background The mosquito A. aegypti is vector of dengue and other viruses. New methods of vector control are needed and can be achieved by a better understanding of the life cycle of this insect. Embryogenesis is a part of A. aegypty life cycle that is poorly understood. In insects in general and in mosquitoes in particular energetic metabolism is well studied during oogenesis, when the oocyte exhibits fast growth, accumulating carbohydrates, lipids and proteins that will meet the regulatory and metabolic needs of the developing embryo. On the other hand, events related with energetic metabolism during A. aegypti embryogenesis are unknown. Results Glucose metabolism was investigated throughout Aedes aegypti (Diptera) embryonic development. Both cellular blastoderm formation (CBf, 5 h after egg laying - HAE) and germ band retraction (GBr, 24 HAE) may be considered landmarks regarding glucose 6-phosphate (G6P) destination. We observed high levels of glucose 6-phosphate dehydrogenase (G6PDH) activity at the very beginning of embryogenesis, which nevertheless decreased up to 5 HAE. This activity is correlated with the need for nucleotide precursors generated by the pentose phosphate pathway (PPP), of which G6PDH is the key enzyme. We suggest the synchronism of egg metabolism with carbohydrate distribution based on the decreasing levels of phosphoenolpyruvate carboxykinase (PEPCK) activity and on the elevation observed in protein content up to 24 HAE. Concomitantly, increasing levels of hexokinase (HK) and pyruvate kinase (PK) activity were observed, and PEPCK reached a peak around 48 HAE. Glycogen synthase kinase (GSK3) activity was also monitored and shown to be inversely correlated with glycogen distribution during embryogenesis. Conclusions The results herein support the hypothesis that glucose metabolic fate changes according to developmental embryonic stages. Germ band retraction is a moment that was characterized as a landmark in glucose metabolism during Aedes aegypti embryogenesis. Furthermore, the results also suggest a role for GSK3 in glycogen balance/distribution during morphological modifications.
Arabinogalactan proteins (AGPs), present in cell walls, plasma membranes and extracellular secretions, are massively glycosylated hydroxyproline-rich proteins that play a key role in several plant developmental processes. After stress treatment, microspores cultured in vitro can reprogramme and change their gametophytic developmental pathways towards embryogenesis, thereby producing embryos which can further give rise to haploid and double haploid plants, important biotechnological tools in plant breeding. Microspore embryogenesis constitutes a convenient system for studying the mechanisms underlying cell reprogramming and embryo formation. In this work, the dynamics of both AGP presence and distribution were studied during pollen development and microspore embryogenesis in Brassica napus, by employing a multidisciplinary approach using monoclonal antibodies for AGPs (LM2, LM6, JIM13, JIM14, MAC207) and analysing the expression pattern of the BnAGP Sta 39-4 gene. Results showed the developmental regulation and defined localization of the studied AGP epitopes during the two microspore developmental pathways, revealing different distribution patterns for AGPs with different antigenic reactivity. AGPs recognized by JIM13, JIM14 and MAC207 antibodies were related to pollen maturation, whereas AGPs labelled by LM2 and LM6 were associated with embryo development. Interestingly, the AGPs labelled by JIM13 and JIM14 were induced with the change of microspore fate. Increases in the expression of the Sta 39-4 gene, JIM13 and JIM14 epitopes found specifically in 2-4 cell stage embryo cell walls, suggested that AGPs are early molecular markers of microspore embryogenesis. Later, LM2 and LM6 antigens increased progressively with embryo development and localized on cell walls and cytoplasmic spots, suggesting an active production and secretion of AGPs during in vitro embryo formation. These results give new insights into the involvement of AGPs as potential regulating/signalling molecules in microspore reprogramming and embryogenesis. PMID:23729197
El-Tantawy, Ahmed-Abdalla; Solís, María-Teresa; Da Costa, Mario L; Coimbra, Silvia; Risueño, María-Carmen; Testillano, Pilar S
In many species, there is little transcription in the mature oocyte, and zygotic transcription does not begin immediately after fertilization. In zebrafish, zygotic transcription is not initiated until the mid-blastula transition, thus the production of new proteins during oogenesis and early embryogenesis is dependent on the translation of maternal mRNAs. In a growing number of species, the translation of key maternal transcripts is coupled to their cytoplasmic polyadenylation. One family of RNA-binding proteins implicated in this process is the cytoplasmic polyadenylation element (CPE)-binding proteins (CPEBs), which bind to a sequence in the 3'-untranslated regions of regulated transcripts and mediate their storage/repression or translation. In several species, there is evidence for two classes of CPEBs, a larger oocyte-type and a smaller CPEB that functions during embryogenesis. This appears to be the case in zebrafish as well, and we now provide evidence suggesting that the oocyte-type CPEB (zorba) regulates the translation of the embryonic-type (ElrA) by keeping the ElrA transcript in storage until fertilization. When zorba levels fall, ElrA protein is then produced and available to regulate the translation of additional mRNAs during embryogenesis. We have also identified a potential target of ElrA, the maternal mRNA for hnRNPab, which is a potential homolog of the Drosophila gene squid, whose product plays a role in patterning the Drosophila oocyte and embryo. These data suggest that during zebrafish embryogenesis, cytoplasmic polyadenylation mediates a cascade of translational control whose final targets play central patterning roles during embryogenesis. PMID:24474627
O'Connell, Marcia L; Cavallo, William C; Firnberg, Maytal
Germline mutations of LKB1/Peutz-Jeghers syndrome gene predispose carriers to hamartomatous polyposis of the gastrointestinal tract as well as to cancer of different organ systems. Although Peutz-Jeghers syndrome patients frequently present with neoplasms of the colon, stomach, small intestine, pancreas, breast, ovaries, and cervix, somatic mutations appear to be rare in the sporadic tumor types thus far studied (colorectal, gastric, testicular, and breast cancers). To evaluate whether somatic mutations of LKB1 contribute to the tumorigenesis of yet unstudied tumor types, we screened 14 cell lines and 129 tumor specimens from different cancers for a genetic defect in LKB1. Six melanoma and eight myeloma cell lines were scrutinized for LKB1 somatic mutations by genomic sequencing. No changes were found in the coding LKB1 sequence and exon/intron boundaries. Next, we analyzed 12 pancreatic, 8 gastric, 12 ovarian granulosa cell, 26 cervical, 28 lung, 24 soft tissue, and 19 renal tumors by single-strand conformational polymorphism analysis. Three changes in LKB1 coding nucleotide sequence were identified. One base pair deletion at A957 and G958 substitution by T occurred in a cervical adenocarcinoma sample, resulting in a frameshift and premature stop codon at position 335. Substitution of A581 by T occurred in a lung adenocarcinoma sample, resulting in the change of aspartic acid at position 194 to valine. A loss of another allele was detected in this sample. One silent change, C1257T, was found in a pancreatic carcinoma sample. The changes were not present in the matched normal tissue DNA samples. Our results suggest that mutational inactivation of LKB1 is a rare event in most sporadic tumor types.
Avizienyte, Egle; Loukola, Anu; Roth, Stina; Hemminki, Akseli; Tarkkanen, Maija; Salovaara, Reijo; Arola, Johanna; Butzow, Ralf; Husgafvel-Pursiainen, Kirsti; Kokkola, Arto; Jarvinen, Heikki; Aaltonen, Lauri A.
Background A key to understanding the evolution of the nervous system on a large phylogenetic scale is the identification of homologous neuronal types. Here, we focus this search on the sensory and motor neurons of bilaterians, exploiting their well-defined molecular signatures in vertebrates. Sensorimotor circuits in vertebrates are of two types: somatic (that sense the environment and respond by shaping bodily motions) and visceral (that sense the interior milieu and respond by regulating vital functions). These circuits differ by a small set of largely dedicated transcriptional determinants: Brn3 is expressed in many somatic sensory neurons, first and second order (among which mechanoreceptors are uniquely marked by the Brn3+/Islet1+/Drgx+ signature), somatic motoneurons uniquely co-express Lhx3/4 and Mnx1, while the vast majority of neurons, sensory and motor, involved in respiration, blood circulation or digestion are molecularly defined by their expression and dependence on the pan-visceral determinant Phox2b. Results We explore the status of the sensorimotor transcriptional code of vertebrates in mollusks, a lophotrochozoa clade that provides a rich repertoire of physiologically identified neurons. In the gastropods Lymnaea stagnalis and Aplysia californica, we show that homologues of Brn3, Drgx, Islet1, Mnx1, Lhx3/4 and Phox2b differentially mark neurons with mechanoreceptive, locomotory and cardiorespiratory functions. Moreover, in the cephalopod Sepia officinalis, we show that Phox2 marks the stellate ganglion (in line with the respiratory — that is, visceral— ancestral role of the mantle, its target organ), while the anterior pedal ganglion, which controls the prehensile and locomotory arms, expresses Mnx. Conclusions Despite considerable divergence in overall neural architecture, a molecular underpinning for the functional allocation of neurons to interactions with the environment or to homeostasis was inherited from the urbilaterian ancestor by contemporary protostomes and deuterostomes.
Conversion of somatic cells to pluripotency by defined factors is a long and complex process that yields embryonic stem cell-like cells that vary in their developmental potential. To improve the quality of resulting induced pluripotent stem cells (iPSCs), which is important for potential therapeutic applications, and to address fundamental questions about control of cell identity, molecular mechanisms of the reprogramming process must be understood. Here we discuss recent discoveries regarding the role of reprogramming factors in remodeling the genome, including new insights into the function of c-Myc, and describe the different phases, markers and emerging models of reprogramming.
Buganim, Yosef; Faddah, Dina A.; Jaenisch, Rudolf
Excised zygotic embryos, mericarps ("seeds") and hypocotyls of seedlings of cultivated carrot Daucus carota cv. Scarlet Nantes were evaluated for their ability to generate somatic embryos on a semisolid hormone-free nutrient medium. Neither intact zygotic embryos nor hypocotyls ever produced somatic embryos. However, mericarps and broken zygotic embryos were excellent sources for somatic embryo production (response levels as high as 86%). Somatic embryo formation was highest from cotyledons, but was also observed on isolated hypocotyls and root tips of mature zygotic embryos. On media containing unreduced nitrogen, somatic embryo formation led to the generation of vigorous cultures comprised entirely of somatic embryos at various stages of development which in turn proliferated still other somatic embryos. However, a medium was devised which when 1-5 mM NH4+ was the sole nitrogen source, led only to a proliferation of globular proembryos. Sustained subculturing of these proembryos at 2-3 week intervals enabled establishment of highly uniform cultures in which no further development into more mature stages of embryonic development occurred. These have been maintained, without decline, as morphogenetically competent proembryonic globules for over ten months. A basal medium containing from 1-5 mM NH4+ as the sole nitrogen source appears not to be inductive to somatic proembryo formation. Instead, such a medium is best thought of as permissive to the expression of embryogenically determined cells within zygotic embryos. By excising and breaking or wounding zygotic embryos, constituent cells are probably released from positional or chemical restraints and thus are able to express their innate embryogenic potential. Once a proembryonic culture is established, this medium containing 1-5 mM NH4+ as the sole nitrogen source provides a nonpermissive environment to the development and growth of later embryonic stages, but it does allow the continued formation and multiplication of globular somatic proembryos. The sequence of events leading from excised broken zygotic embryos to the formation of somatic embryos and the maintenance of somatic proembryos are demonstrated by scanning electron microscopy and histological preparations. Germination levels from intact zygotic embryos on media with varying levels and ratios of unreduced vs. reduced inorganic nitrogen were determined as well and provided baseline or control data on the type of response obtained from nonwounded material.
Smith, D. L.; Krikorian, A. D.
The nitrate-reductase (NR) defective cell lines of Nicotiana plumbaginifolia isolated in our laboratory could not be regenerated into plants on the standard medium (Márton et al. 1982 a). The normal regeneration potential, however, was restored in somatic hybrids obtained by fusing the NR(-) (green) lines with a pigment deficient (P(-)), but NR(+) line, A28. Somatic hybrid plants were fertile in two combinations (A28 + NA9 and A28 + NX9). As expected, segregation for NR(-) and P(-) was found after selfing the somatic F1 (SF1) obtained by protoplast fusion, and in the F2. The variable segregation ratios are explained by chromosome abnormalities. Co-segregation of the NR(-) phenotype and the altered response to shoot induction on standard medium suggest the involvement of the nitrate-assimilatory pathway in determining shoot regeneration ability. PMID:24253002
Márton, L; Biasini, G; Maliga, P
Somatic mutation theory explains how DNA damage can lead to the malignant transformation of cells. It therefore elucidates the connection between genotoxic agents and cancers. Mutational spectra, which tend to be characteristic of a cancer type, are available for certain genes like p53 which is frequently mutated in tumors. A mutational spectrum could therefore be the signature of the genotoxic agent(s) at the origin of the malignant transformation. Ligation-mediated PCR (LMPCR) is a genomic sequencing method that can be used for the mapping of DNA damage at nucleotide resolution. Such a mapping can then be compared to a mutational spectrum to test the hypothesis that implies one agent can cause mutations into one cancer type. LMPCR has been used this way to map DNA damage generated by different UV wavelengths. The frequently damaged sites following UVB irradiation correlate with the mutational spectrum of p53 in skin cancer. Similarly, BPDE, the activated form of the benzo[a]pyrene present in tobacco smoke, generates frequent adducts at sites corresponding to mutation hotspots of p53 in lung cancers. Still, the correlation between BPDE damage sites and p53 mutations is not perfect and this suggests a role of other genotoxic substances that are also present in tobacco smoke, such as the nitrosamine NNK. Finally, and beyond this objective of better understanding somatic mutagenesis, LMPCR is commonly used whenever DNA damage frequency and/or repair is to be investigated. PMID:20188052
Lacoste, Sandrine; Rochette, Patrick J; Drouin, Régen
Differentiation of adult bone marrow stem cells (BMSC) into hepatocyte-like cells is commonly performed by continuous exposure to a cytokines-cocktail. Here, it is shown that the differentiation efficacy in vitro can be considerably enhanced by sequential addition of liver-specific factors (fibroblast growth factor-4, hepatocyte growth factor, insulin-transferrin-sodium selenite, and dexamethasone) in a time-dependent order that closely resembles the secretion pattern during in vivo liver embryogenesis. Quantitative RT-PCR analysis and immunocytochemistry showed that, upon sequential exposure to liver-specific factors, different stages of hepatocyte differentiation, as seen during liver embryogenesis, can be mimicked. Indeed, expression of the early hepatocyte markers alpha-fetoprotein and hepatocyte nuclear factor (HNF)3beta decreased as differentiation progressed, whereas levels of the late liver-specific markers albumin (ALB), cytokeratin (CK)18, and HNF1alpha were gradually upregulated. In contrast, cocktail treatment did not significantly alter the expression pattern of the hepatic markers. Moreover, sequentially exposed cells featured highly differentiated hepatic functions, including ALB secretion, glycogen storage, urea production, and inducible cytochrome P450-dependent activity, far more efficiently compared to the cocktail condition. In conclusion, sequential induction of the differentiation process, analogous to in vivo liver development, is crucial for in vitro differentiation of adult rat BMSC into functional hepatocyte-like cells. This model may not only be applicable for in vitro studies of endoderm differentiation but it also provides a "virtually unlimited" source of functional hepatocytes, suitable for preclinical pharmacological research and testing, and cell and organ development. PMID:16840566
Snykers, Sarah; Vanhaecke, Tamara; Papeleu, Peggy; Luttun, Aernout; Jiang, Yuehua; Vander Heyden, Yvan; Verfaillie, Catherine; Rogiers, Vera
The purpose of the present study on canine somatic cell nuclear transfer (SCNT) was to evaluate the effects of fusion strength, type of activation, culture media and site of transfer on developmental potential of SCNT embryos. We also examined the potential of enucleated bovine oocytes to serve as cytoplast recipients of canine somatic cells. Firstly, we evaluated the morphological characteristics
G. Jang; H. J. Oh; M. K. Kim; Y. H. Fibrianto; M. S. Hossein; H. J. Kim; J. J. Kim; S. G. Hong; J. E. Park; S. K. Kang; B. C. Lee
Purpose: Of all nonauditory sensory systems, only the somatosensory system seems to be related to tinnitus (eg, temporomandibular joint syndrome and whiplash). The purpose of this study is to describe the distinguishing characteristics of tinnitus associated with somatic events and to use these characteristics to develop a neurological model of somatic tinnitus.Materials and Methods: Case series.Results: Some patients with tinnitus,
Robert Aaron Levine
There is renewed interest in using animal oocytes to reprogram human somatic cells. Here we compare the re- programming of human somatic nuclei using oocytes obtained from animal and human sources. Comparative analysis of gene expression in morula-stage embryos was carried out using single-embryo transcriptome am- plification and global gene expression analyses. Genomic DNA fingerprinting and PCR analysis confirmed that
Young Chung; Colin E. Bishop; Nathan R. Treff; Stephen J. Walker; Vladislav M. Sandler; Sandy Becker; Irina Klimanskaya; Wan-Song Wun; Randall Dunn; Rebecca M. Hall; Jing Su; Shi-Jiang Lu; Marc Maserati; Young-Ho Choi; Richard Scott; Anthony Atala; Ralph Dittman; Robert Lanza
Since the 1970s, somatics have increasingly become a part of the dance training landscape. Although the psychophysical benefits seem sufficient in themselves to warrant inclusion in dance, this article explores another possible outcome of embracing somatic pedagogical principles, a change that affects not "what" is taught in a dance class, but…
Restriction site polymorphisms between two parental mitochondrial (mt) genomes were used to characterize the novel mt genome present in a somatic hybrid plant. A cosmid clone containing mtDNA restriction fragments characteristic of both parental plant lines was identified in a library constructed from mtDNA of progeny of a somatic hybrid plant. Restriction mapping revealed the location of several restriction fragments
Madge Rothenberg; Maury L. Boeshore; Maureen R. Hanson; Shamay Izhar
Somatic mutation calling from next-generation sequencing data remains a challenge due to the difficulties of distinguishing true somatic events from artifacts arising from PCR, sequencing errors or mis-mapping. Tumor cellularity or purity, sub-clonality and copy number changes also confound the identification of true somatic events against a background of germline variants. We have developed a heuristic strategy and software (http://www.qcmg.org/bioinformatics/qsnp/) for somatic mutation calling in samples with low tumor content and we show the superior sensitivity and precision of our approach using a previously sequenced cell line, a series of tumor/normal admixtures, and 3,253 putative somatic SNVs verified on an orthogonal platform.
Kassahn, Karin S.; Holmes, Oliver; Nones, Katia; Patch, Ann-Marie; Miller, David K.; Christ, Angelika N.; Harliwong, Ivon; Bruxner, Timothy J.; Xu, Qinying; Anderson, Matthew; Wood, Scott; Leonard, Conrad; Taylor, Darrin; Newell, Felicity; Song, Sarah; Idrisoglu, Senel; Nourse, Craig; Nourbakhsh, Ehsan; Manning, Suzanne; Wani, Shivangi; Steptoe, Anita; Pajic, Marina; Cowley, Mark J.; Pinese, Mark; Chang, David K.; Gill, Anthony J.; Johns, Amber L.; Wu, Jianmin; Wilson, Peter J.; Fink, Lynn; Biankin, Andrew V.; Waddell, Nicola; Grimmond, Sean M.; Pearson, John V.
Summary Identification of somatic rearrangements in cancer genomes has accelerated through analysis of high-throughput sequencing data. However, characterization of complex structural alterations and their underlying mechanisms remains inadequate. Here, applying an algorithm to predict structural variations from short reads, we report a comprehensive catalog of somatic structural variations and the mechanisms generating them, using high-coverage whole-genome sequencing data from 140 patients across ten tumor types. We characterize the relative contributions of different types of rearrangements and their mutational mechanisms, find that ~20% of the somatic deletions are complex deletions formed by replication errors, and describe the differences between the mutational mechanisms in somatic and germline alterations. Importantly, we provide detailed reconstructions of the events responsible for loss of CDKN2A/B and gain of EGFR in glioblastoma, revealing that these alterations can result from multiple mechanisms even in a single genome and that both DNA double-strand breaks and replication errors drive somatic rearrangements.
Yang, Lixing; Luquette, Lovelace J.; Gehlenborg, Nils; Xi, Ruibin; Haseley, Psalm S.; Hsieh, Chih-Heng; Zhang, Chengsheng; Ren, Xiaojia; Protopopov, Alexei; Chin, Lynda; Kucherlapati, Raju; Lee, Charles; Park, Peter J.
In this study, we assessed five types of childhood trauma (i.e., physical, sexual, and emotional abuses; the witnessing of violence; physical neglect) and their relationship to somatic preoccupation in adulthood. Using a cross-sectional sample of convenience, we surveyed 113 individuals who were being seen as outpatients in an internal medicine setting and seeking non-emergent medical care. With yes/no response options, we inquired about five types of childhood trauma (i.e., physical, sexual, and emotional abuses; the witnessing of violence; physical neglect) and measured somatic preoccupation with the Bradford Somatic Inventory, a self-report measure. In this study, both physical and emotional abuses demonstrated significant correlations with scores on the Bradford Somatic Inventory. These findings suggest that physical and emotional abuses in childhood may have some relationship with somatic preoccupation in adulthood. PMID:24916826
Sansone, Randy A; Wiederman, Michael W; Tahir, Nighat A; Buckner, Victoria R
In microspore cultures of Brassica napus L. cv. Topas, embryo yield increases with culture density up to about 40,000 microspores per ml. A much higher density (100,000 per ml) appears inhibitory to embryogenesis. A relatively high culture density (30,000 or 40,000 per ml) for the first 2–4 days of culture is crucial for embryogenesis, after which cultures may be diluted
Bin Huang; Sharon Bird; Roger Kemble; Daina Simmonds; Wilf Keller; Brian Miki
Background Large-scale tumor sequencing projects are now underway to identify genetic mutations that drive tumor initiation and development. Most studies take a gene-based approach to identifying driver mutations, highlighting genes mutated in a large percentage of tumor samples as those likely to contain driver mutations. However, this gene-based approach usually does not consider the position of the mutation within the gene or the functional context the position of the mutation provides. Here we introduce a novel method for mapping mutations to distinct protein domains, not just individual genes, in which they occur, thus providing the functional context for how the mutation contributes to