Gostaríamos de agradecer Tal Nawy e Martin Bayer para os seus conselhos sobre o isolamento do embrião. MTR, VG, UG e CB inventou o equipamento de isolamento de embriões. MTR, VG e CB desenvolveu o protocolo de isolamento de embriões. MTR, VG e CB estabelecido o protocolo, isolado dos embriões, e embrião gerado cDNA, VG realizada a PCR, MTR a coloração GUS, JJ experimentos os peixes. MTR, VG, CG e UG escreveu o manuscrito. Este trabalho foi financiado pela Universidade de Zurique, uma bolsa da Fundação Roche Research (a MTR), e doações da Fundação Nacional da Suíça (para UG e CB).

O procedimento está resumido no diagrama de fluxo mostrado na Figura 1. O microcapilares e a configuração instrumentais são apresentados na Figura 2 e Figura 3, e os passos típicos de isolamento de embriões são mostrados na Figura 4. 1. Material e Preparação de buffer 1,1 Silício revestimento de vidro microcapilares <ol><li> Coloque a microcapilares em um tubo Falcon de 15 ml com ~ 5 ml de Sigmacote (Sigma) e gire várias vezes. </li><li> Remover a solução, colocar um tubo Falcon contendo os capilares numa folha de alumínio e cozer durante 3 horas a 60 ° C. Guarde à temperatura ambiente. </li></ol> 1.2 Obter ~ 50-100 micrometros de diâmetro dicas microcapilares <ol><li> Puxe um milímetro de diâmetro capilares de vidro manualmente ao longo de um bico de Bunsen ou usando um extrator comercial (vertical puxador de filamento ou micropipeta extrator). </li><li> Use um diamantecaneta de ponta ou lâmina para cortar a extremidade do capilar puxado para criar a abertura desejada. Selecione a melhor forma de capilares sob um microscópio estéreo. A abertura deve ser de 50-100 mM (Figura 2). </li></ol> 1.3 Preparação da lâmina <ol><li> Siliconize lâminas limpas, cobrindo todos os poços com Sigmacote (~ 1 ml / lâmina), durante 5 minutos, remover. Asse 3 hr em papel alumínio. Guarde à temperatura ambiente. </li><li> Lavar os múltiplos poços lâminas microscópicas de vidro durante 10 min em SDS a 10%, 2 x 2 minutos em água livre de nuclease (autoclavado DEPC ddH2O), 2 min em etanol a 70%, 2 minutos em etanol a 100%, com ar- secar. Todas as etapas são feitas em frascos autoclavados Coplin. As lâminas podem ser reutilizados várias vezes proporcionando limpeza completa entre cada uso. </li><li> Pouco antes do isolamento propagação embrião ~ 0,5 l de 10 mg / ml de albumina de soro bovino (BSA), com uma pipeta ao longo de toda a superfície de cada poço e ar seco. </li></ol> 1.4 Microscópio e configuração capilar &lt;ol&gt; <li> Use um microscópio invertido com uma objetiva de 20x e 10x. Otimizar o contraste de luz (embriões são bastante transparente). </li><li> Coloque um micromanipulador para segurar o capilar de vidro, ao lado do microscópio. O capilar de vidro, está ligado a um microinjector (Figura 3A) ou para um P-200 pipeta regularmente através de um tubo de borracha (Figura 3B, ver a discussão para uma descrição detalhada desta configuração). </li><li> Colocar o tubo capilar acima da lâmina de microscópio com um ângulo de ~ 70 ° (figura 3) e ajustar a posição de ter a abertura no campo de visão. </li><li> Pouco antes do isolamento do embrião tomar-se e libertar ~ 5-10 ul de BSA (1 mg / ml) para revestir o capilar. </li></ol> 1,5 Buffers A Tabela 1 lista os buffers de isolamento e de destino, dependendo das aplicações a jusante. <ol><li> Prepare ~ 1 ml de tampão de isolamento por amostra imediatamente antes da utilização e mantenha-o no gelo. </li><li> Prepare o buffer de destino em um ml tubo de baixa ligação 0,5 Eppendorf no gelo (aplicações moleculares) ou em uma lâmina de microscópio (aplicações citológicas) em uma câmara úmida. </li></ol> 2. Dissecção sementes e extração de embriões 2.1 Sincronização de desenvolvimento da semente <ol><li class="jove_content"> Emasculate flores e mantê-los 2 dias em câmara de crescimento, evitando o contacto dos pistilos expostas com outras flores, então polinizar deles (por exemplo, 11). </li><li class="jove_content"> Teste o estágio de desenvolvimento em suas condições de crescimento por meio de investigação microscópica de sementes limpas. Com as nossas condições de crescimento (16 horas de luz a 21 ° C, 8 horas escuro, a 18 ° C e 70% de humidade), as sementes recolhidas 2,5 dias após a polinização (DAP) rendeu principalmente 2-4 embriões de células e as sementes colhidas 3,5-4 DAP rendeu globular embriões. </li></ol> 2.2 Dissecção de Sementes e Rupture <ol><li> Remover a sEEDS 10-15 siliques (~ 2,5 DAP) sob um microscópio estereoscópico com uma pinça e agulhas de insulina. </li><li> Mergulhar as sementes em 20 ul de tampão de isolamento, em 2 ml de um tubo de fundo redondo de Eppendorf colocada em gelo. </li><li> Suavemente esmagar as sementes com um pilão de plástico (pré-lavado com 10% de SDS, enxaguado com DEPC ddH2O e lavou-se com etanol a 70%) para libertar os embriões até o extracto de semente é turvo. A força a aplicar é determinada pelo utilizador, a cada ensaio. </li><li> Lavar o almofariz com 300 ul de tampão de isolamento para lavar o pilão e diluir a amostra. </li><li> Spin-se o extrato a 5.000 xg por 5 seg. Delicadamente ressuspender o extrato sedimentados por pipetagem cima e para baixo 2-3 vezes. </li><li> Filtrar o extracto com uma malha de nylon de 30 um (montado em adaptadores de tubos, por exemplo, a partir de PartecCelltricks). Lavar a malha com um tampão de isolamento adicional de 200 ul. </li></ol> 3. Isolamento embrião 3.1 Preparação da lâmina <ol> &lt;li&gt; Coloque um pano limpo, multi-slides bem BSA-revestido e siliconizada no palco do microscópio invertido, ressuspender o extrato de semente de filtrada com cuidado pipetando cima e para baixo e pipetar 2 gotas de extrato de semente de 40-50 mL em um ou dois poços . Screening apenas 1 ou 2 gotas de cada vez evita a evaporação da amostra. </li><li> Transferem-se 50 ul de tampão de isolamento fresco (1 gota de lavagem r) num poço de uma lâmina diferente preparada como antes. Manter este slide em uma coberta, câmara úmida para evitar a evaporação. </li></ol> 3.2 tela, limpar, recolher <ol><li> Tela de gotas de extrato de semente de embriões no estágio desejado com o objetivo de ampliação de 10x. Se necessário, confirmar o palco com a ampliação de 20x. Os embriões geralmente descem para o fundo do slide. </li><li> Remover manualmente os restos em torno do embrião com uma agulha de tungstênio, uma agulha de insulina ou equipamento similar. </li><li> Mover o vidro capilar próximo do embrião utilizando o micromanipulador, take-se o embrião com tão pouco quanto possível solução. </li><li> Colete vários embriões (por exemplo, todos uma gota) e liberá-los no gota de lavagem 1 º (aplicação molecular) ou no buffer de destino (aplicações citológicos). Cada rodada coleta deve ser mantido dentro de 5-10 min e embriões devem ser coletadas em um volume mínimo (o volume total de todas as rodadas de coleta deve ser &lt;5 mL). </li><li> Repita a triagem e recolha até a quantidade desejada de embriões é recolhida no r gota uma lavagem (centralmente, se possível, para facilitar a recordação). </li><li> Recordar todos os embriões de uma só vez a partir da queda de lavagem (se os restos são transportados, retire-os com uma agulha antes de recordação). </li><li> Solte os embriões em uma gota de lavagem 2 º de 50 l. Repita 3.2.6. </li><li> Solte os embriões no buffer de destino. A transferência deve envolver apenas um contato, e não de imersão, da ponta do capilar. </li><li> Substitua o microcapilLary para a próxima amostra. </li></ol>

<ol>
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L., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+demethylation+of+repetitive+elements+during+seed+development+underlies+gene+imprinting.">Extensive demethylation of repetitive elements during seed development underlies gene imprinting.</a> Science. 324, 1447-1451 (2009).</li><li>Muller, B., Sheen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokinin+and+auxin+interaction+in+root+stem-cell+specification+during+early+embryogenesis.">Cytokinin and auxin interaction in root stem-cell specification during early embryogenesis.</a> Nature. 453, 1094-1097 (2008).</li><li>Gehring, M., Missirian, V., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+analysis+of+parent-of-origin+allelic+expression+in+Arabidopsis+thaliana+seeds.">Genomic analysis of parent-of-origin allelic expression in Arabidopsis thaliana seeds.</a> PLoS One. 6, 23687-23 (2011).</li><li>Xiang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+reveals+gene+expression+and+metabolic+network+dynamics+during+embryo+development+in+Arabidopsis.">Genome-wide analysis reveals gene expression and metabolic network dynamics during embryo development in Arabidopsis.</a> Plant Physiol. 156, 346-356 (2011).</li><li>Nawy, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GATA+factor+HANABA+TARANU+is+required+to+position+the+proembryo+boundary+in+the+early+Arabidopsis+embryo.">The GATA factor HANABA TARANU is required to position the proembryo boundary in the early Arabidopsis embryo.</a> Dev Cell. 19, 103-113 (2010).</li><li>Baroux, C., Pecinka, A., Fuchs, J., Schubert, I., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+triploid+endosperm+genome+of+Arabidopsis+adopts+a+peculiar,+parental-dosage-dependent+chromatin+organization.">The triploid endosperm genome of Arabidopsis adopts a peculiar, parental-dosage-dependent chromatin organization.</a> Plant Cell. 19, 1782-1794 (2007).</li><li>Autran, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+epigenetic+pathways+control+parental+contributions+to+Arabidopsis+early+embryogenesis.">Maternal epigenetic pathways control parental contributions to Arabidopsis early embryogenesis.</a> Cell. 145, 707-719 (2011).</li><li>Wohrmann, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+DNA+methylation-independent+imprinting+control+region+at+the+Arabidopsis+MEDEA+locus.">Identification of a DNA methylation-independent imprinting control region at the Arabidopsis MEDEA locus.</a> Genes Dev. 26, 1837-1850 (2012).</li><li>Raissig, M. T., Baroux, C., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+Flexibility+of+Genomic+Imprinting+during+Seed+Development.">Regulation and Flexibility of Genomic Imprinting during Seed Development.</a> Plant Cell. 23, 16-26 (2011).</li><li>Rea, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+DNA+methylation+of+imprinted+genes+in+Arabidopsis+endosperm.">Determination of DNA methylation of imprinted genes in Arabidopsis endosperm.</a> J. Vis Exp. 47 (47), e2327 (2011).</li><li>Breuninger, H., Rikirsch, E., Hermann, M., Ueda, M., Laux, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+WOX+genes+mediates+apical-basal+axis+formation+in+the+Arabidopsis+embryo.">Differential expression of WOX genes mediates apical-basal axis formation in the Arabidopsis embryo.</a> Dev Cell. 14, 867-876 (2008).</li><li>Kohler, C., Page, D. R., Gagliardini, V., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+thaliana+MEDEA+Polycomb+group+protein+controls+expression+of+PHERES1+by+parental+imprinting.">The Arabidopsis thaliana MEDEA Polycomb group protein controls expression of PHERES1 by parental imprinting.</a> Nature. 37, 28-30 (2005).</li><li>Fransz, P., De Jong, J. H., Lysak, M., Castiglione, M. R., Schubert, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interphase+chromosomes+in+Arabidopsis+are+organized+as+well+defined+chromocenters+from+which+euchromatin+loops+emanate.">Interphase chromosomes in Arabidopsis are organized as well defined chromocenters from which euchromatin loops emanate.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 14584-14589 (2002).</li><li>Morris, J., Singh, J. M., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+analysis+of+single+cells.">Transcriptome analysis of single cells.</a> J. Vis. Exp. (50), e2634 (2011).</li><li>Nodine, M. D., Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+and+paternal+genomes+contribute+equally+to+the+transcriptome+of+early+plant+embryos.">Maternal and paternal genomes contribute equally to the transcriptome of early plant embryos.</a> Nature. 482 (7383), (2012).</li><li>Jefferson, R. A., Kavanagh, T. A., Bevan, M. W. G. U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fusions:+beta-glucuronidase+as+a+sensitive+and+versatile+gene+fusion+marker+in+higher+plants.">fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.</a> EMBO J. 6, 3901-3907 (1987).</li><li>Schmidt, A., W&#246;hrmann, H., Raissig, M. T., Arand, J., Gheyselinck, J., Gagliardini, V., Heichinger, C., Walter, J., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Polycomb+group+protein+MEDEA+and+the+DNA+methyltransferase+MET1+interact+in+Arabidopsis+to+repress+autonomous+endosperm+development.">The Polycomb group protein MEDEA and the DNA methyltransferase MET1 interact in Arabidopsis to repress autonomous endosperm development.</a> Plant J. 73 (5), (1111).</li></ol>

Os autores declaram que não têm interesses financeiros concorrentes.

Foi desenvolvido um protocolo de isolamento do embrião, que é rápida, eficaz, e pode ser facilmente transferido para outros laboratórios. O equipamento aqui descrito é composto por um microscópio invertido, um micromanipulador, vidro microcapilares, um puxador de filamento vertical e um microinjector (Figura 3A). A instalação é semelhante à descrita para o isolamento única célula animal para análises transcriptomics 17. Também trabalhou com sucesso c...

O embrião de plantas floridas está rodeado pelo endosperma, um tecido nutritivo derivado de um segundo evento de fertilização. Ambos embrião e endosperma está cercado por várias camadas de células do tegumento. Colectivamente estes tecidos formar uma semente, que se desenvolvem no interior do fruto. Assim, tecidos e análises específicas de células de embriões de Arabidopsis são fortemente prejudicada devido a sua inacessibilidade. No entanto, os embriões nas fases de fim de globulares ou mais tarde, são relativamente bem passível de dissecção manual utilizando agulhas finas de tungstênio sob o microscópio estereoscópico, ou com uma ligeira pressão sobre a semente, usando uma pinça para extraí-los. Tais técnicas foram utilizadas com sucesso para transcriptoma ou análises de perfis epigenoma como hibridização microarray, sequenciamento bissulfito, ou RNA seqüência (por exemplo, 1-3). Em contraste, os estudos de embriões no zigoto até a fase globular cedo permanecem tecnicamente desafiador. Até o momento, poucos estudos têm repanálises de transcriptoma orted sobre jovens embriões usando tanto microdissection de captura a laser (LCM) de tecidos embrionários de sementes seções fixas quatro ou extração manual dos embriões individuais a partir de sementes, utilizando ferramentas finas 5. No entanto, o equipamento GCV não é vulgarmente disponíveis e extracção manual do embrião em fases iniciais é demorado e que exige excelentes capacidades de dissecação que não são facilmente transferíveis. Em adição à análise de todo o genoma, em análises de expressão de genes in situ também são difíceis de executar em jovens, conjunto de montagem em embriões de Arabidopsis. Em certa medida, os embriões jovens pode ser libertado em lâminas de microscópio por uma suave pressão sobre as sementes e utilizada para os ensaios de gene repórter ou de detecção de proteínas por coloração imunológica (por exemplo, ver 6,7). Esta técnica, no entanto, não permitir o isolamento de embriões de alto rendimento, impedindo assim as análises quantitativas. Por isso, desenvolvemos um protocolo eficiente e rápidapara o isolamento de embrião a partir de sementes de Arabidopsis que é simples de configurar, facilmente transferíveis, e apropriado para uma variedade de aplicações a jusante. O princípio básico consiste em triturar suavemente sementes - dissecada jovens siliques em um tubo Eppendorf utilizando um pilão de plástico num tampão de isolamento adequado. O extracto de semente é colocada em gotas sobre uma lâmina de multi-poços e é pesquisado quanto à presença de embriões libertados na fase desejada usando um microscópio invertido. Os embriões são coletados por meio de um vidro microcapilar ligado a um microinjetor ou uma pipeta de laboratório padrão. Para aplicações moleculares, os embriões são lavados duas vezes por libertação repetida em gotas de novo tampão de isolamento antes de transferi-los para o buffer de destino num volume mínimo. Para aplicações citológicos (ensaios repórter, positividade, peixe), as etapas de lavagem pode ser omitido. O método oferece várias vantagens: (i) produz 25-40 embriões em ~ 45 min para aplicação citológicocações ou em 3-4 hr para aplicações moleculares (incluindo as etapas de lavagem), (ii) que permite o isolamento de estágios embrionários específicas, (iii) é facilmente transferível para outras pessoas e laboratórios, devido à sua configuração simples, (iv) requer equipamento acessível para a configuração básica, que é passível de upgrades, e (v) foi usado com sucesso por várias aplicações a jusante, como RNA seqüenciamento 8, a análise de DNA-metilação-específica gene 9, ensaios repórter (10 e Raissig et al., em prep.) e FISH (J. Jaenisch, U. Grossniklaus, C. Baroux inédito, ver Figura 5).

<table border="1">
	<tbody>
 <tr>
 <td>Name of the Reagent</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>Sigmacote</td>
 <td>SIGMA </td>
 <td>SL2-100 ml</td>
 <td></td>
 </tr>
 <tr>
 <td>RNAse OUT</td>
 <td>Invitrogen (life technologies)</td>
 <td>10777-019</td>
 <td></td>
 </tr>
 <tr>
 <td>First- strand buffer</td>
 <td>Invitrogen (life technologies)</td>
 <td>18064-022</td>
 <td>contained in Superscript II package</td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>Invitrogen (life technologies)</td>
 <td>18064-022</td>
 <td>contained in Superscript II package</td>
 </tr>
 <tr>
 <td>Bovine serum albumin (BSA) 100x =10 mg/ml </td>
 <td>New England Biolabs Inc.</td>
 <td></td>
 <td>Different suppliers will also work </td>
 </tr>
 <tr>
 <td>Thin wall Capillaries 1.0 mm</td>
 <td>World Precision Instruments</td>
 <td>TW100F-4</td>
 <td></td>
 </tr>
 <tr>
 <td>DNA LoBind tube 0.5 ml</td>
 <td>Vaudaux-Eppendorf</td>
 <td>0030108.035</td>
 <td></td>
 </tr>
 <tr>
 <td>CellTrics&Delta; 30 &mu;m</td>
 <td>PARTEC</td>
 <td>04-0042-2316</td>
 <td></td>
 </tr>
 <tr>
 <td>5wells 10 mm diameter slides</td>
 <td>Electron Microscopy Sciences</td>
 <td>63421-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Formaldehyde Solution</td>
 <td>Sigma-Aldrich</td>
 <td>F1635</td>
 <td></td>
 </tr>
 <tr>
 <td>Superfrost Plus slide</td>
 <td>Thermo Fisher</td>
 <td>J1800AMNZ</td>
 <td>Menzel-Gl&auml;ser</td>
 </tr>
 <tr>
 <td>Tris</td>
 <td>Amaresco</td>
 <td>0497</td>
 <td></td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Applichem</td>
 <td>A2937</td>
 <td></td>
 </tr>
 <tr>
 <td>Glycin</td>
 <td>Fluka</td>
 <td>50050</td>
 <td></td>
 </tr>
 <tr>
 <td>SDS pellets</td>
 <td>Roth</td>
 <td>CN30.3</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro Pestle</td>
 <td>VWR</td>
 <td>431-0094</td>
 <td></td>
 </tr>
 <tr>
 <td>Microfine insulin syringes</td>
 <td>BD</td>
 <td>U-100</td>
 <td></td>
 </tr>
 <tr>
 <td>DEPC</td>
 <td>Sigma-Aldrich</td>
 <td>D5758</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Schaurlau</td>
 <td>ET00102500</td>
 <td></td>
 </tr>
 <tr>
 <td>Forceps N5</td>
 <td>Dumont</td>
 <td>0108-5</td>
 <td></td>
 </tr>
 <tr>
 <td>Bioanalyzer Pico Chip</td>
 <td>Agilent Technologies</td>
 <td>5067-1513</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Inverted microscope</td>
 <td>Nikon TMS (Japan),</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Micromanipulator</td>
 <td>Leitz</td>
 <td></td>
 <td>Leica</td>
 </tr>
 <tr>
 <td>Micomanipulator Post mount LH1 probe</td>
 <td>Leica microsystems</td>
 <td>39430101</td>
 <td>Different brand will also do the work</td>
 </tr>
 <tr>
 <td>Vertical filament puller</td>
 <td>Sutter instrument</td>
 <td>P-20 model</td>
 <td>Other model are also suitable </td>
 </tr>
 <tr>
 <td>Cell Tram vario</td>
 <td>Vaudaux-Eppendorf</td>
 <td>5176.000.033</td>
 <td></td>
 </tr>
 <tr>
 <td>Bioanalyzer</td>
 <td>Agilent Technologies</td>
 <td>2100</td>
 <td></td>
 </tr>
 <tr>
 <td>Qubit Fluorometer</td>
 <td>Invitrogen (life technologies</td>
 <td></td>
 <td></td>
 </tr>
	 </tbody>
 </table>

efficient and rapid isolation of early-stage embryos from arabidopsis thaliana seeds

Em plantas com flores, o embrião se desenvolve dentro de um tecido nutritivo - o endosperma - cercado pelos tegumentos de sementes maternas (ou tegumento). Como consequência, o isolamento de embriões de plantas em fases iniciais (1 célula para a fase globular) é tecnicamente difícil devido à sua relativa inacessibilidade. Dissecção Manual eficiente em fases iniciais é fortemente prejudicada devido ao pequeno tamanho dos jovens de sementes de Arabidopsis e a adesividade do embrião para os tecidos circundantes. Aqui, nós descrevemos um método que permite o isolamento eficiente de embriões jovens de Arabidopsis, originando até 40 embriões em 1 hora e 4 horas, dependendo da aplicação a jusante. Os embriões são libertados no tampão de isolamento por esmagamento ligeiramente 250-750 sementes com um pilão de plástico num tubo Eppendorf. Um copo microcapilar ligado a qualquer uma pipeta padrão de laboratório (através de um tubo de borracha) ou de um microinjetor controlada hidraulicamente é usado para coletar embriões de lugar gotículasd numa lâmina de multi-poços em um microscópio óptico invertido. As competências técnicas necessárias são simples e facilmente transferível, ea configuração básica não requer equipamento caro. Os embriões recolhidos são apropriados para uma variedade de aplicações a jusante, tais como RT-PCR, o RNA de sequenciação, análise de metilação de ADN, hibridação in situ fluorescente (FISH), a imunocoloração, e ensaios de gene repórter.

O processo de isolamento do embrião (Figura 1) permite o isolamento de até 40 embriões em 4 horas, se as lavagens são realizadas, por exemplo, para aplicações moleculares, ou em menos de uma hora, se as lavagens são omitidos, por exemplo, para aplicações citológicos. Figura 2 exibe alta e baixa dicas microcapilares qualidade e A Figura 3 mostra a configuração da máquina de isolamento do embrião. Figura 4 mostra o processo...

Watch this Scientific Journal Video about Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds at JoVE.com

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

Nós relatamos um método eficiente e simples para isolar os embriões em estágios iniciais de desenvolvimento de<em&gt; Arabidopsis thaliana</em&gt; Sementes. Até 40 embriões podem ser isolados em 1 hora a 4 horas, dependendo da aplicação a jusante. O procedimento é adequado para transcriptoma, a metilação do DNA, a expressão do gene repórter, imunomarcação e fluorescência<em&gt; In situ</em&gt; Análises de hibridização.

Isolamento eficiente e rápida de embriões em início de carreira a partir de<em&gt; Arabidopsis thaliana</em&gt; Sementes

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

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Biology

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

17.7K visualizações.  University of Zürich. Nós relatamos um método eficiente e simples para isolar os embriões em estágios iniciais de desenvolvimento de Arabidopsis thaliana Sementes. Até 40 embriões podem ser isolados em 1 hora a 4 horas, dependendo da aplicação a jusante. O procedimento é adequado para transcriptoma, a metilação do DNA, a expressão do gene repórter, imunomarcação e fluorescência In situ Análises de hibridização.

Vídeo: Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

Fator de Crescimento colocando-Revestido Beads em embriões de galinha Early Stage

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

Biologia

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

Isolamento de Protoplastos de tecidos de 14 dias de idade, mudas de Arabidopsis thaliana</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

Floral-dip Transformação de Arabidopsis thaliana Examine a PTSO2:: β-glucuronidase Expressão Gênica Reporter

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

Arabidopsis thaliana Profiling Glycerolipid Polar por cromatografia em camada delgada (TLC) Juntamente com Cromatografia gás-líquido (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Ensaio para imunoprecipitação da cromatina Genes Tissue-específicos usando embriões em início de carreira do rato

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Isolamento e Purificação de cinesina partir Drosophila Os embriões

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Geração rápida e eficiente de neurônios a partir de células-tronco pluripotentes humanas, em um formato de placa Multititre

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Metabólica Labeling e Membrana Fracionamento para Comparative Analysis proteômica de<em&gt; Arabidopsis thaliana</em&gt; Culturas suspensão de células

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

Coloração histoquímica de<em&gt; Arabidopsis thaliana</em&gt; Celular Secundária parede Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

Método padronizado para a esterilização da elevado-produção de sementes de Arabidopsis

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Isolamento eficiente e rápida de embriões em início de carreira a partir de

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