Global scale transcriptome analysis of Arabidopsis embryogenesis in vitro

Background Somatic embryogenesis (SE) in plants is a process by which embryos are generated directly from somatic cells, rather than from the fused products of male and female gametes. Despite the detailed expression analysis of several somatic-to-embryonic marker genes, a comprehensive understanding of SE at a molecular level is still lacking. The present study was designed to generate high resolution transcriptome datasets for early SE providing the way for future research to understand the underlying molecular mechanisms that regulate this process. We sequenced Arabidopsis thaliana somatic embryos collected from three distinct developmental time-points (5, 10 and 15 d after in vitro culture) using the Illumina HiSeq 2000 platform. Results This study yielded a total of 426,001,826 sequence reads mapped to 26,520 genes in the A. thaliana reference genome. Analysis of embryonic cultures after 5 and 10 d showed differential expression of 1,195 genes; these included 778 genes that were more highly expressed after 5 d as compared to 10 d. Moreover, 1,718 genes were differentially expressed in embryonic cultures between 10 and 15 d. Our data also showed at least eight different expression patterns during early SE; the majority of genes are transcriptionally more active in embryos after 5 d. Comparison of transcriptomes derived from somatic embryos and leaf tissues revealed that at least 4,951 genes are transcriptionally more active in embryos than in the leaf; increased expression of genes involved in DNA cytosine methylation and histone deacetylation were noted in embryogenic tissues. In silico expression analysis based on microarray data found that approximately 5% of these genes are transcriptionally more active in somatic embryos than in actively dividing callus and non-dividing leaf tissues. Moreover, this identified 49 genes expressed at a higher level in somatic embryos than in other tissues. This included several genes with unknown function, as well as others related to oxidative and osmotic stress, and auxin signalling. Conclusions The transcriptome information provided here will form the foundation for future research on genetic and epigenetic control of plant embryogenesis at a molecular level. In follow-up studies, these data could be used to construct a regulatory network for SE; the genes more highly expressed in somatic embryos than in vegetative tissues can be considered as potential candidates to validate these networks.

Transformação genética em cevada por bombardeamento de partículas

A presente Tese de Doutorado objetivou: (1) definir um método eficiente de transformação genética, por bombardeamento de partículas, para a obtenção de plantas transgênicas de cultivares brasileiras de cevada e (2) identificar gene(s) codificante(s) de quitinase(s) potencialmente capaz(es) de conferir resistência ao fungo patogênico de cevada Bipolaris sorokiniana. Culturas de calos obtidos a partir de escutelos imaturos das cultivares Brasileiras de cevada MN-599 e MN-698 (Cia. de Bebidas das Américas, AMBEV) foram bombardeadas com partículas de tungstênio e avaliadas quanto à expressão do gene repórter gusA através de ensaios histoquímicos de GUS e quanto ao efeito dos bombardeamentos na indução estruturas embriogênicas e regeneração de plantas. As condições de biobalística analisadas incluíram a região promotora regulando a expressão de gusA, tipo e pressão de gás hélio de dois aparelhos de bombardeamento, distância de migração das partículas, número de tiros e a realização de pré e pós-tratamento osmótico dos tecidos-alvo. No presente trabalho foram obtidos um número bastante alto de pontos azuis por calo, a indução de calos embriogênicos e embriões somáticos em uma freqüência de até 58,3% e a regeneração de 60 plantas, sendo 43 de calos bombardeados. As melhores condições observadas foram o promotor e primeiro íntron do gene Adh de milho (plasmídeo pNGI), o aparelho de bombardeamento “ Particle Inflow Gun” (PIG) utilizando-se a distância de migração de partículas de 14,8 cm, dois tiros disparados por placa e a realização de tratamento osmótico dos explantes com 0,2 M de manitol e 0,2 M de sorbitol 4-5 horas antes e 17-19 horas depois dos bombardeamentos. Das 43 plantas obtidas de calos bombardeadas, 3 apresentaram atividade de GUS na base das suas folhas. A utilização de primers sintéticos definidos a partir de genes de quitinases descritos na literatura em PCRs resultou na amplificação de dois fragmentos de aproximadamente 700 e 500 pb a partir de DNA total das cvs. MN-599 e MN-698 de cevada e um fragmento, com aproximadamente 500 pb, a partir do DNA total do isolado A4c de Trichoderma sp. Estes fragmentos foram purificados dos géis de agarose e diretamente seqüenciados de forma manual e automática. Os fragmentos de 700 e 500 pb amplificados do genoma da cultivar MN-599 foram identificados como genes de quitinases de cevada e o fragmento de 500 pb do isolado A4c de Trichoderma sp. não apresentou homologia com seqüências conhecidas de quitinases depositadas no EMBL/GenBank. A utilização de novos pares de primers, representando seqüências conservadas de quitinases do fungo Metarhizium anisopliae, resultou na amplificação de 3 fragmentos a partir do DNA total do isolado A4b de Trichoderma sp., que estão sendo purificados para realização de seqüenciamento.

Avaliação da eficiência de co-transformação de soja (Glycine max (L.) Merrill) via bombardeamento de partículas utilizando um gene de seleção e um gene de interesse em plamídeos diferentes

Visando aumentar a resistência a moléstias fúngicas, o presente trabalho teve como objetivo introduzir um gene (chit1) que codifica uma quitinase do fungo Metarhizium anisopliae em cultivares de soja [Glycine max (L.) Merrill]. A co-transformação foi a estratégia escolhida, visando a obtenção de plantas livres de transgenes marcadores na progênie das plantas transformadas. A co-transformação foi realizada via biolística, tendo como tecido-alvo conjuntos de embriões somáticos globulares das cultivares MG/BR46 Conquista e IAS-5. O plasmídeo pGusHyg, que contém o gene repórter gusA e o gene marcador hpt, foi bombardeado concomitantemente com o plasmídeo pMOG463chit1, que porta o gene chit1. Os conjuntos de embriões bombardeados foram transferidos para meio seletivo contendo higromicina, visando a obtenção de material estavelmente transformado. Os conjuntos embriogênicos higromicina-resistentes foram transferidos seqüencialmente para meios de proliferação D-20 (sem higromicina), maturação e regeneração. No total, foram obtidos 387 e 380 embriões histodiferenciados das cultivares MG/BR46 Conquista e IAS-5, respectivamente. Plantas transgênicas adultas e férteis foram regeneradas. Para avaliar a eficiência da estratégia de cotransformação, foram realizadas análises moleculares de embriões histodiferenciados e de plantas regeneradas. Os resultados obtidos neste trabalho permitiram o cálculo da taxa de co-transformação de 44% para os embriões histodiferenciados da cultivar MG/BR46 Conquista e de 50% para plantas de IAS-5. Não existem, até o momento, relatos de trabalhos em soja utilizando embriões somáticos globulares em proliferação como alvo para estudos de co-transformação.

Indução de embriogênese somática em cupuaçu (Theobroma grandiflorum Schum.)

Folhas jovens de cupuaçu (Theobroma grandiflorum Schum.) foram empregadas para indução de embriogênese somática, as quais foram cultivadas inicialmente em meio MS suplementado com 6,0 mg/L BAP e 0,5 mg/L AIA (meio de estabelecimento), seguindo-se o cultivo em meio MS suplementado com 2,20 mg/L TDZ (meio de indução). A região da nervura mostrou intumescimento três dias após inoculação no meio de estabelecimento, o qual foi seguido pela formação de cachos de calos. As massas calosas foram transferidas para o meio de indução, e as estruturas com características pró-embriogênicas puderam ser observadas após uma semana. Estudos de microscopia eletrônica de varredura revelaram estruturas com características de embriões somáticos no meio com TDZ.

Capacidade embriogênica da cultivar IAS-5 de soja

O objetivo deste trabalho foi estudar o controle genético da formação de embriões somáticos da cultivar IAS-5 de soja. O experimento foi conduzido em casa de vegetação, cultivando-se quatro plantas por vaso, sob fotoperíodo de 14 horas e temperatura em torno de 28°C. Efetuaram-se cruzamentos entre os parentais não-embriogênicos (cultivares IAC-6, Paraná e IAC-15) e embriogênico (cultivar IAS-5) e retrocruzamentos para obtenção das gerações F1, F2,RC1P1 e RC1P2. Cotilédones imaturos, com 4-6 mm de comprimento, derivados dos parentais das gerações F1, F2, RC1P1 e RC1P2 foram cultivados em placas de Petri contendo meio N10, por um período de 90 dias, em câmara de crescimento. Os embriões somáticos derivados da indução foram contados, e os números, usados para obtenção dos parâmetros genéticos. Os resultados obtidos mostraram que o caráter capacidade de produção de embriões somáticos da cultivar IAS-5 é de natureza quantitativa e controlado por, aproximadamente, 20 genes.

Indução de calos em espécies amazônicas do gênero Theobroma

Vários trabalhos vem sendo desenvolvidos sobre o cultivo in vitro de cacau (T.cacao), mas são raros para a maioria das outras espécies do gênero, como o cupuaçu (T. grandiflorum), cuja a área plantada vem aumentando expressivamente, e outras que poderiam servir de fonte de genes para as espécies economicamente já reconhecidas. Protocolos para obtenção de embriões somáticos in vitro para as espécies T. cacao,T. grandiflorum,T. speciosum e o híbrido T. grandiflorum x T. obovatum foram avaliados a partir de duas fontes de explantes, estaminódios e pétalas (formadas por lígulas e cógulas) cultivados em meio de crescimento primário de calo, consistindo de sais DKW, suplementado com 20 g l^-1 de sacarose, 250 mg l^-1de glutamina, 200 mg l^-1 de mio-inositol, 0,2 mg l^-1 de tiamina-HCl, 0,1 mg l^-1 de ácido nicotínico, 0,2 mg l^-1 de glicina, 2 mg l^-1 de 2,4-D, 2,2 g l^-1 de Gelrite® e pH 5,8. A este meio foram adicionadas diferentes concentrações de tidiazuron (0, 5 e 10 µg l^-1). As culturas foram mantidas no escuro por 14 dias, à temperatura de 25 ± 2 ºC, e então transferidas para meio de crescimento secundário de calo, constituído de sais WPM, vitaminas de Gamborg, 20 g l^-1 de sacarose, 2 mg l^-1 de 2,4 D, 0,3 mg l^-1 de cinetina, 50 ml l^-1 de água de côco, 2,2 g l^-1 de Gelrite® e pH 5,8. A formação de calos ocorreu em todas as espécies. Embriões somáticos foram obtidos somente para T. cacao. A calogênese mostrou-se influenciada pelo genótipo e foi maior nos estaminódios.

In vitro conservation of Dendrobium germplasm

Dendrobium is a large genus in the family Orchidaceae that exhibits vast diversity in floral characteristics, which is of considerable importance to orchid breeders, biotechnologists and collectors. Native species have high value as a result of their medicinal properties, while their hybrids are important as ornamental commodities, either as cut flowers or potted plants and are thus veritable industrial crops. Thus, preservation of Dendrobium germplasm is valuable for species conservation, breeding programs and the floriculture industry. Cryopreservation represents the only safe, efficient and cost-effective long-term storage option to facilitate the conservation of genetic resources of plant species. This review highlights 16 years of literature related to the preservation of Dendrobium germplasm and comprises the most comprehensive assessment of thorough studies performed to date, which shows reliable and reproducible results. Air-drying, encapsulation-dehydration, encapsulation-vitrification, vitrification and droplet-vitrification are the current cryopreservation methodologies that have been used to cryopreserve Dendrobium germplasm. Mature seeds, pollen, protoplasts, shoot primordia, protocorms and somatic embryos or protocorm-like bodies (PLBs) have been cryopreserved with different levels of success. Encapsulation-vitrification and encapsulation-dehydration are the most used protocol, while PLBs represent the main explant explored.

Funktionelle Untersuchungen von FLORICAULA und KNOX-Genen in Eschscholzia californica Cham.

FLORICAULA (FLO) und KNOTTED1-like Homöobox (KNOX)-Gene übernehmen neben ihren konservierten Funktionen in der Achsenentwicklung in verschiedenen Eudikotylen eine Funktion in der Fiederblattentwicklung. Zur Klärung der Frage nach dem ursprünglichen Regulationsweg der Fiederblattentwicklung in Hinblick auf FLO und KNOX-Gene innerhalb der Eudikotylen wurde hier die Bedeutung dieser Gene für die Fiederblattentwicklung von Eschscholzia californica als Modell für die Ranunculales, die Schwestergruppe aller anderen Eudikotylen untersucht. Es wurde ein Protokoll zur Erzeugung von somatischen Embryonen aus unreifen Samen entwickelt. Wege zur Herstellung von Mutanten durch Agrobacterium-vermittelte Transformation werden vorgeschlagen. Die Bedeutung von Auxin für die Blattentwicklung und die Untersuchung der Interaktion von ESCHSCHOLZIA CALIFORNICA FLORICAULA (EcFLO) und des KNOX- Gens ESCHSCHOLZIA CALIFORNICA SHOOT MERISTEMLESS (EcSTM) mit Auxin wurde durch Hemmung des Auxintransports untersucht. Trotz gravierender Störungen in der Blattpositionierung und -morphologie konnten Expressionsänderungen beider Gene nicht nachgewiesen werden. Ein Funktionsverlust von EcFLO und KNOX-Genen in E. californica wurden mittels Virus induziertem Gen Silencing (VIGS) erzeugt. VIGS von EcFLO rief keinen Phänotypen hervor. VIGS des KNOX-Gens EcSTM erzeugte dagegen in einigen Pflanzen eine Reduktion der Fiederzahl. Auch molekularbiologisch konnte das Silencing von EcSTM, nicht aber das Silencing von EcFLO nachgewiesen werden. Die Ergebnisse belegen die Notwendigkeit des ungestörten Auxintransports für die Blattentwicklung von E. californica und machen die Beteiligung des KNOX-Gens EcSTM an der Blattentwicklung wahrscheinlich. Die Beteiligung von EcFLO an der Fiederbildung konnte nicht nachgewiesen werden.

Obtención de clones de alcornoque (Quercus suber L.) mediante embriogénesis somática: aplicación de medios líquidos y biorreactores en la sincronización de los procesos de desarrollo y maduración de los embriones

El alcornoque tiene un gran valor ambiental, como integrante de los ecosistemas forestales mediterráneos, e interés comercial por el valor de la bellota (alimentación del cerdo ibérico), el carbón, la madera y sobre todo por las aplicaciones industriales del corcho. Las posibilidades de mejora genética del alcornoque, como las de otras especies forestales, están limitadas por sus largos ciclos reproductivos y porque su propagación vegetativa mediante estaquillado solo es posible en estados muy juveniles. Por ello este sistema de propagación tiene muy poca, o ninguna, utilidad práctica en la mejora genética. La embriogénesis somática es la vía más apropiada para la clonación de muchas especies forestales y ha hecho posible el desarrollo a gran escala de plantaciones multivarietales de coníferas. En alcornoque es posible la regeneración completa de árboles adultos mediante embriogénesis somática. Con los protocolos actuales (en medio semisólido), los embriones se generan formando acúmulos y en la fase de multiplicación conviven embriones en distintos estados de desarrollo. Es un sistema asincrónico, con baja eficacia para la propagación en masa, que no elimina completamente las dificultades para el desarrollo de programas de mejora genética del alcornoque. En otras especies la utilización de medios líquidos ha mejorado: la sincronización, productividad de los cultivos, el manejo y reducido los costes de producción. Por ello el desarrollo de suspensiones embriogénicas de alcornoque se plantea como una vía para aumentar la eficacia de la propagación clonal a gran escala. En la presente tesis se desarrollan cultivos embriogénicos de alcornoque en medio líquido. El capítulo 3 aborda el establecimiento y mantenimiento de suspensiones, el capítulo 4 el desarrollo de una fase de proliferación en medio líquido y el capítulo 5 la utilización de sistemas de cultivo en medio líquido, estacionarios y de inmersión temporal, como vía para favorecer la maduración de los embriones somáticos. Para iniciar los cultivos en medio líquido se emplearon agregados de embriones tomados de la fase de proliferación en medio semisólido. Cuando estos agregados se inocularon directamente en medio líquido no se logró el establecimiento de las suspensiones. El establecimiento se consiguió empleando como inóculo las células y Resumen pequeños agregados embriogénicos, de tamaño comprendido entre 41 y 800 μm, desprendidas por agitación breve de los agregados de embriones. El mantenimiento se logró inoculando en baja densidad masas embriogénicas compactas de tamaño comprendido entre 0,8 y 1,2 mm. Estas suspensiones, muy heterogéneas, mantuvieron su capacidad de proliferación y de regeneración de embriones al menos durante diez subcultivos consecutivos. El protocolo de iniciación y mantenimiento, desarrollado inicialmente con un solo genotipo, fue eficaz cuando se probó sobre otros 11 genotipos de alcornoque. En la fase de proliferación se ensayaron tres tipos de envase y tres velocidades de agitación. La combinación envase × velocidad determinó el intercambio gaseoso, la disponibilidad de oxígeno y el estrés hidrodinámico. Los agregados embriogénicos de alcornoque crecieron incluso en condiciones de hipoxia no siendo la disponibilidad de oxígeno un factor limitante del crecimiento para tasas de trasferencia de oxígeno comprendidas entre 0,11 h-1 y 1,47 h-1. Por otra parte la producción de biomasa creció con el estrés hidrodinámico para valores de índice de cizalladura inferiores a 5 x 10-3 cm min-1. La mayor producción de biomasa se obtuvo con matraces Erlenmeyer de 100 ml y alta velocidad de agitación (160 rpm) mientras que la diferenciación de embriones se vio favorecida por bajas velocidades de agitación (60 rpm) asociadas con bajas disponibilidades de oxígeno. La posibilidad de madurar embriones de alcornoque en medio líquido se estudió utilizando sistemas de inmersión permanente y sistemas de inmersión temporal. En inmersión permanente no se diferenciaron embriones cotiledonares (posiblemente por hiperhidricidad). Los sistemas de inmersión temporal permitieron obtener embriones maduros en estado cotiledonar y capaces de regenerar plantas in vitro. Concentraciones de sacarosa superiores a 60 g l-1 y frecuencias de inmersión iguales o inferiores a una diaria, tuvieron efectos negativos para el desarrollo de los embriones somáticos. En los sistemas de inmersión temporal los parámetros físico-químicos del medio de cultivo se mantuvieron estables y no se observó ninguna limitación de nutrientes. No obstante, estos sistemas se vieron afectados por la evaporación que generó el flujo de aire necesario para desplazar el líquido en cada periodo de inmersión. Abstract ABSTRACT Cork oak is one of the most important tree species of the Mediterranean ecosystem. Besides its high environmental value has a great economic interest due to the sustainable production of acorns (to feed the Iberian pig) charcoal, timber and cork, which is a renewable natural product with various technological applications. As happens with other forest species, cork oak genetic improvement programs are limited by their long life cycles and because vegetative propagation by cuttings it´s only possible in very juvenile plants. Hence this propagation system is useless or has little practical use for breeding cork oak. Plant regeneration by somatic embryogenesis is the most suitable way for cloning many forest species, and it is the enabling technology which has allowed the establishment of large-scale conifer multi-varietal plantations. Clonal plant regeneration of mature cork oak trees can be achieved through somatic embryogenesis. Somatic embryos at different stages of development and forming clusters are produced during the multiplication phase with current protocols (using semisolid medium). This is an asynchronous low-efficient process not suitable for mass propagation, and therefore it does not solve the difficulties presented by cork oak breeding programs. Culture in liquid medium has been used with other species to improve: synchronization, yield, handling, and to reduce production costs. Thus the development of cork oak embryogenic suspension cultures is envisaged as a way to increase the efficiency of large scale clonal propagation. The thesis herein develops cork oak embryogenic cultures in liquid medium. In chapter 3 establishment and maintenance of suspension cultures are developed, chapter 4 studies proliferation phase in liquid medium and chapter 5 considers the use of different systems of culture in liquid medium, both stationary and temporary immersion, as a way to promote somatic embryos maturation. Clusters of embryos taken from proliferating cultures on semisolid medium were used to initiate the cultures in liquid medium. When these clusters were inoculated directly in liquid medium establishment of suspension cultures was not executed. However using, as initial inoculum, cells and cell aggregates with a size between 41 and 800 μm detached from these clusters of embryos, subjected to a brief shaking, suspension cultures could be established. Suspension maintenance was achieved by inoculating compact embryogenic Abstract clumps with a size between 0.8 and 1.2 mm at low density. The suspension cultures, very heterogeneous, retained both their proliferation and embryo regeneration capacity for at least ten consecutive subcultures. The initiation and maintenance protocol, initially developed with a single genotype, was effective when tested on 11 additional genotypes of cork oak. In proliferation phase three types of vessels and three different levels of agitation were assayed. The combination vessel × orbiting speed determined gas exchange, oxygen availability and hydrodynamic stress. Cork oak embryogenic aggregates grew even under hypoxia conditions; oxygen availability at transfer rates between 0.11 and 1.47 h-1 was not a limiting factor for growth. Furthermore the biomass production was increased with hydrodynamic stress when shear rate values were of less than 5 x 10-3 cm min-1. The highest biomass production was obtained with 100 ml Erlenmeyer flask and high stirring speed (160 rpm) while the differentiation of embryos was favored by low agitation speeds (60 rpm) associated with low oxygen availability. The possibility to mature cork oak somatic embryos in liquid medium was studied using both permanent immersion systems and temporary immersion systems. Cotyledonary embryos did not differentiate in permanent immersion conditions (probably due to hyperhydricity). Temporary immersion systems allowed obtaining mature cotyledonary embryos, which were able to regenerate plants in vitro. Sucrose concentrations above 60 g l-1 and immersion frequencies equal to or lower than one each 24 h had negative effects on somatic embryo development. Physicochemical parameters of the culture medium in temporary immersion systems were stable and showed no limitation of nutrients. However, these systems were affected by the evaporation generated by the airflow necessary to relocate the medium at each immersion period.

Expression Pattern of the Carrot EP3 Endochitinase Genes in Suspension Cultures and in Developing Seeds1

Carrot (Daucus carota) extracellular protein 3 (EP3) class IV endochitinases were previously identified based on their ability to rescue somatic embryos of the temperature-sensitive cell line ts11. Whole-mount in situ hybridization revealed that a subset of the morphologically distinguishable cell types in embryogenic and nonembryogenic suspension cultures, including ts11, express EP3 genes. No expression was found in somatic embryos. In carrot plants EP3 genes are expressed in the inner integumentary cells of young fruits and in a specific subset of cells located in the middle of the endosperm of mature seeds. No expression was found in zygotic embryos. These results support the hypothesis that the EP3 endochitinase has a “nursing” function during zygotic embryogenesis and that this function can be mimicked by suspension cells during somatic embryogenesis.

Effect of physical, chemical and light treatments on germination and growth of tissue-cultured coconuts

The acclimatization and ex vitro establishment of tissue cultured coconut plantlets regenerated either from zygotic or somatic embryos could result to serious losses. Although high germination rates can be achieved in vitro, the survival of zygotic embryo derived plantlets in soil is very low (0-30%). Hence, treatments that could promote development of good quality seedlings having well-developed shoot and root is needed to increase seedling survival ex vitro. The effect of physical, chemical and light quality treatments on germination and growth of coconut embryos and tissue-cultured seedlings respectively, was investigated. The germination of coconut embryos was promoted when placed in a liquid Euwens (Y3) medium and incubated using a roller drum. Gibberellic acid (GA3) significantly affected growth of seedlings as it promoted shoot elongation, shoot and root expansion, and fresh and dry weight increase. However, GA3 did not significantly affect germination. In addition, the blue, red and yellow light significantly affected growth of seedlings as it promoted leaf and shoot elongation, fresh and dry weight increase, and root and leaf production. These conditions could be used to improve the growth and survival ex vitro of tissue cultured coconuts.

Impairments in embryonic genome activation in rhesus monkey somatic cell nuclear transfer embryos

Somatic cell nuclear transfer (SCNT) is a remarkable process in which a somatic cell nucleus is acted upon by the ooplasm via mechanisms that today remain unknown. Here we show the developmental competence (% blastocyst) of embryos derived from SCNT (21%)

The kinetics of donor cell mtDNA in embryonic and somatic donor cell-derived bovine embryos

The mechanisms controlling the outcome of donor cell-derived mitochondrial DNA (mtDNA) in cloned animals remain largely unknown. This research was designed to investigate the kinetics of somatic and embryonic mtDNA in reconstructed bovine embryos during preimplantation development, as well as in cloned animals. The experiment involved two different procedures of embryo reconstruction and their evaluation at five distinct phases of embryo development to measure the proportion of donor cell mtDNA (Bos indicus), as well as the segregation of this mtDNA during cleavage. The ratio of donor cell (B. indicus) to host oocyte (B. taurus) mtDNA (heteroplasmy) from blastomere- (NT-B) and fibroblast- (NT-F) reconstructed embryos was estimated using an allele-specific PCR with fluorochrome-stained specific primers in each sampled blastomere, in whole blastocysts, and in the tissues of a fibroblast-derived newborn clone. NT-B zygotes and blastocysts show similar levels of heteroplasmy (11.0% and 14.0%, respectively), despite a significant decrease at the 9-16 cell stage (5.8%; p < 0.05). Heteroplasmy levels in NT-F reconstructed zygotes, however, increased from an initial low level (4.7%), to 12.9% (p < 0.05) at the 9-16 cell stage. The NT-F blastocysts contained low levels of heteroplasmy (2.2%) and no somatic-derived mtDNA was detected in the gametes or the tissues of the newborn calf cloned. These results suggest that, in contrast to the mtDNA of blastomeres, that of somatic cells either undergoes replication or escapes degradation during cleavage, although it is degraded later after the blastocyst stage or lost during somatic development, as revealed by the lack of donor cell mtDNA at birth.

Aberrant expression of E-cadherin and beta-catenin proteins in placenta of bovine embryos derived from somatic cell nuclear transfer

Abnormal placental development is common in the bovine somatic cell nuclear transfer (SCNT)-derived fetus. In the present study, we characterised the expression of E-cadherin and beta-catenin, structural proteins of adherens junctions, in SCNT gestations as a model for impaired placentation. Cotyledonary tissues were separated from pregnant uteri of SCNT (n - 6) and control pregnancies (n - 8) obtained by artificial insemination. Samples were analysed by western blot, quantitative RT-PCR (qRT-PCR) and immunohistochemistry. Bovine trophectoderm cell lines derived from SCNT and control embryos were analysed to compare with the in utero condition. Although no differences in E-cadherin or beta-catenin mRNA abundance were observed in fetal tissues between the two groups, proteins encoded by these genes were markedly under-expressed in SCNT trophoblast cells. Immunohistochemistry revealed a different pattern of E-cadherin and total beta-catenin localisation in SCNT placentas compared with controls. No difference was observed in subcellular localisation of dephosphorylated active-beta-catenin protein in SCNT tissues compared with controls. However, qRT-PCR confirmed that the wingless (WNT)/beta-catenin signalling pathway target genes CCND1, CLDN1 and MSX1 were downregulated in SCNT placentas. No differences were detected between two groups of bovine trophectoderm cell lines. Our results suggest that impaired expression of E-cadherin and beta-catenin proteins, along with defective beta-catenin signalling during embryo attachment, specifically during placentation, is a molecular mechanism explaining insufficient placentation in the bovine SCNT-derived fetus.