Wir möchten Tal Nawy und Martin Bayer für ihre Beratung auf den Embryo Isolierung danken. MTR, VG, UG und CB entwickelt der Embryo Isolierung Ausrüstung. MTR, VG und CB entwickelt der Embryo Isolation Protokoll. MTR, VG und CB etablierte das Protokoll, die Embryonen isoliert und erzeugten Embryo-cDNA durchgeführt VG die PCR, MTR die GUS-Färbung, JJ die FISH Experimente. MTR, VG, CG und UG schrieb das Manuskript. Diese Arbeit wurde von der Universität Zürich, ein Stipendium der Roche Research Foundation (MTR) und Zuschüsse aus dem Schweizerischen Nationalfonds (UG und CB) finanziert.

Das Verfahren ist in dem Flussdiagramm in 1 gezeigt zusammengefasst. Die Mikrokapillaren und die instrumentale Setup sind in Abbildung 2 und Abbildung 3 dargestellt und typische Schritte Embryo Isolierung sind in Abbildung 4 dargestellt. 1. Material-und Buffer Vorbereitung 1.1 Silicon Beschichtung von Glas Mikrokapillaren <ol><li> Legen Sie die Mikrokapillaren in einem 15 ml Falcon-Röhrchen mit ~ 5 ml Sigmacote (Sigma) und invertieren mehrmals. </li><li> Entfernen Sie die Lösung, platzieren Sie den Falcon Röhrchen mit der Kapillaren in einer Aluminiumfolie und backen Sie sie für 3 Stunden bei 60 ° C Lagerung bei Raumtemperatur. </li></ol> 1.2 Erhalten ~ 50-100 um Durchmesser mikrokapillaren Tipps <ol><li> Ziehen 1 mm Durchmesser Glaskapillaren entweder manuell über einen Bunsenbrenner oder durch Verwendung eines handelsüblichen Abzieher (vertikale Faden Abzieher oder Feinpipettenziehvorrichtung). </li><li> Verwenden Sie eine Diamant-Filzstift oder Klinge, um die Spitze der Kapillare gezogen geschnitten, um die gewünschte Öffnung zu schaffen. Wählen Sie die beste-förmigen Kapillaren unter einem Stereomikroskop. Die Öffnung sollte 50-100 um (Abbildung 2). </li></ol> 1.3 Slide Vorbereitung <ol><li> Siliconize sauber Schlitten durch die alle Vertiefungen mit Sigmacote (~ 1 ml / Folie) für 5 min, zu entfernen. Bake 3 Stunden in Alufolie. Lagerung bei Raumtemperatur. </li><li> Waschen Sie die Multi-Well-Glas Objektträger für 10 min in 10% SDS, 2 x 2 min in Nuklease-freiem Wasser (DEPC autoklaviert-ddH 2 O), 2 min in 70% Ethanol, 2 min in 100% Ethanol, Luft- trocknen. Alle Schritte werden in autoklavierte Coplin Gläser getan. Folien können mehrmals verwendet werden, sofern eine gründliche Reinigung zwischen jedem Gebrauch. </li><li> Unmittelbar vor dem Embryo isoliert Ausbreitung ~ 0,5 ul 10 mg / ml Rinderserumalbumin (BSA) mit einer Pipette über die gesamte Oberfläche jeder Vertiefung und an der Luft trocknen. </li></ol> 1.4 Mikroskop und Kapillar-Setup &lt;ol&gt; <li> Mit einem inversen Mikroskop mit 10x und 20x Vergrößerung Ziel. Optimieren Sie das Licht Kontrast (Embryonen sind sehr transparent). </li><li> Legen Sie eine Mikromanipulators die Glaskapillare neben dem Mikroskop zu halten. Die Glaskapillare mit einem Mikroinjektor (3A) oder einem regelmäßigen P-200 Pipette über einen Gummischlauch verbunden (Fig. 3B, siehe die Diskussion für eine ausführliche Beschreibung des Setup). </li><li> Legen Sie die Kapillare über dem Objektträger bei einer ~ 70 ° Winkel (Abbildung 3) und passen Sie die Position, um die Öffnung in das Blickfeld zu haben. </li><li> Kurz vor dem Embryo Isolation aufnehmen und abgeben ~ 5-10 ul BSA (1 mg / ml) zur Beschichtung der Kapillare. </li></ol> 1.5 Puffer Tabelle 1 listet die Isolierung und Zielpuffer Abhängigkeit von den Downstream-Anwendungen. <ol><li> Bereiten ~ 1 ml Isolation Puffer pro Probe vor Gebrauch frisch und bewahren Sie es auf Eis. </li><li> Bereiten Sie die Ziel-Puffer in einem 0,5 ml Eppendorf schwach bindende Röhrchen auf Eis (molekulare Anwendungen) oder auf einem Objektträger (zytologische Anwendungen) in einer feuchten Kammer. </li></ol> 2. Seed Dissection und Embryo Extraction 2.1 Synchronisation von Seed Entwicklung <ol><li class="jove_content"> Emasculate Blumen und halten sie 2 Tage im Wachstum der Kammer, während die Vermeidung von Kontakt der exponierten pistils mit anderen Blumen, dann bestäuben sie (z. B. 11). </li><li class="jove_content"> Testen Sie die Stufe der Entwicklung unter den Wachstumsbedingungen durch mikroskopische Untersuchung von Saatgut gelöscht. Mit unserem Wachstumsbedingungen (16 Stunden Licht bei 21 ° C, 8 Stunden Dunkelheit bei 18 ° C und 70% Luftfeuchtigkeit) Samen gesammelt 2,5 Tage nach der Bestäubung (DAP) lieferte hauptsächlich 2-4 Embryonen und Samen gesammelt 3,5-4 DAP ergab Kugelsternhaufen Embryonen. </li></ol> 2.2 Seed Dissection und Rupture <ol><li> Entfernen Sie die seeds 10-15 Schoten (~ 2,5 DAP) unter einem Stereomikroskop mit einer Pinzette und Insulin Nadeln. </li><li> Tauchen Sie die Samen in 20 ul Isolation Puffer in einem 2-ml-Eppendorf-Röhrchen auf Eis gelegt. </li><li> Vorsichtig zerdrücken Sie die Samen mit einer Kunststoff-Stößel (mit 10% SDS Pre-gereinigt, gespült mit DEPC-ddH2O gewaschen und mit 70% Ethanol), um die Embryonen freizugeben, bis die Samen-Extrakt ist bewölkt. Die Kraft anzuwenden ist, die von jedem Benutzer bei Versuch bestimmt werden. </li><li> Spülen Sie den Stößel mit 300 ul Puffer, um die Isolation Stößel waschen und Verdünnen Sie die Probe. </li><li> Spin-down der Extrakt bei 5.000 xg für 5 Sekunden. Vorsichtig resuspendieren pelletiert Extrakt durch Pipettieren up-and-down 2-3 mal. </li><li> Der Extrakt wird mit einer 30 um Nylon-Mesh (montiert auf Rohr-Adapter, zB von PartecCelltricks). Spülen Sie die Masche mit einer zusätzlichen Isolierung 200 ul Puffer. </li></ol> 3. Embryo Isolation 3.1 Slide Vorbereitung <ol> &lt;li&gt; Ein sauberes, BSA-beschichteten und silikonisierten Multi-Well-Schieber auf der Bühne des inversen Mikroskop, suspendieren die gefilterte Extrakt durch Pipettieren vorsichtig nach oben und unten und Pipette 2 Tropfen von 40-50 ul-Extrakt in 1 oder 2 Brunnen . Screening nur 1 bis 2 Tropfen auf einmal verhindert die Verdunstung der Probe. </li><li> Platz 50 ml frisches Isolation Puffer (1. wash &amp; Drop) in eine Vertiefung einer anderen Folie wie zuvor vorbereitet. Bewahren Sie diese Folie in einem überdachten, feuchten Kammer, um Verdunstung zu verhindern. </li></ol> 3.2 Bildschirm, reinigen, sammeln <ol><li> Bildschirm Der Tropfen Extrakt für Embryonen in der gewünschten Stufe mit dem 10-facher Vergrößerung Ziel. Bestätigen Sie ggf. die Bühne mit dem 20-facher Vergrößerung. Die Embryonen in der Regel an der Unterseite des Schlittens sinken. </li><li> Manuell entfernen Sie Schmutz rund um den Embryo mit einer Wolfram-Nadel, einer Insulin-Nadel oder ähnlichen Geräten. </li><li> Bewegen Sie die Glaskapillare in der Nähe der Embryo mit dem Mikromanipulators take up des Embryos mit so wenig wie möglich Lösung. </li><li> Sammle mehrere Embryonen (z. B. alle ein Tröpfchen) und lassen sie in der 1. Wäsche Tropfen (molekulare Anwendung) oder in Ziel-Puffer (zytologische Anwendungen). Jede Runde sammeln sollte innerhalb von 5-10 min und Embryonen aufbewahrt werden sollten in einem minimalen Volumen (das Gesamtvolumen aller Sammeln Runden sollte &lt;5 ul sein) gesammelt werden. </li><li> Wiederholen Sie das Screening und Sammlung, bis die gewünschte Menge an Embryonen in der 1. Wäsche Tropfen gesammelt (zentral, wenn möglich, um Erinnerung zu erleichtern). </li><li> Erinnern Sie alle Embryonen auf einmal aus der Wäsche Tropfen (wenn Schutt durchgeführt werden über, entfernen Sie sie mit einer Nadel vor Erinnerung). </li><li> Lassen Sie die Embryonen in einem 2. waschen Tropfen von 50 pl. Wiederholen 3.2.6. </li><li> Lassen Sie die Embryonen in der Ziel-Puffer. Die Übertragung sollte beinhalten nur einen Kontakt, und nicht Eintauchen der Kapillare Spitze. </li><li> Ersetzen Sie die microcapillARY für die nächste Probe. </li></ol>

<ol>
	<li>Gehring, M., Bubb, K. 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>

Die Autoren erklären, dass sie keine finanziellen Interessen haben.

Wir entwickelten einen Embryo Isolation Protokoll, das eine schnelle, effektive ist, und kann leicht an andere Labors übertragen werden. Die beschriebenen Geräte besteht hier aus einem inversen Mikroskop, einem Mikromanipulator, Glas-Mikrokapillaren ein vertikaler Faden Abzieher und Mikroinjektor (Abbildung 3A). Das Setup ist ähnlich dem für Einzeltier Zellisolation für Transkriptom-Analysen 17 beschrieben. Wir haben auch erfolgreich mit einem Basis-Setup, wo...

Der Embryo von Blütenpflanzen wird durch das Endosperm, ein Nährgewebe von einer zweiten Düngung Veranstaltung abgeleitet umgeben. Sowohl Embryo und Endosperm durch mehrere Zellschichten der Samenschale umgeben. Gemeinsam bilden diese Gewebe einen Samen, der in der Frucht zu entwickeln. So werden Gewebe-und Zell-spezifische Analysen von Arabidopsis Embryonen stark beeinträchtigt durch ihre Unzugänglichkeit. Dennoch sind Embryonen bei den späten Kugelsternhaufen oder späteren Phasen relativ gut zugänglich manuelle Dissektion mit feinen Wolfram Nadeln unter dem Stereomikroskop, oder durch leichten Druck auf das Saatgut mit einer Pinzette, um sie zu extrahieren. Solche Techniken wurden erfolgreich für Transkriptom oder epigenome Profiling Analysen wie Microarray-Hybridisierung, Bisulfit-Sequenzierung oder RNA-Sequenzierung (zB 1-3) verwendet. Im Gegensatz dazu bleiben Studien von Embryonen bei der Zygote bis Anfang globulären Stadium technisch anspruchsvoll. Bisher haben nur wenige Studien reported Transkriptomanalysen auf junge Embryonen entweder mit Laser-Mikrodissektion (LCM) von embryonalen Geweben von festen Samen Abschnitten 4 oder manuelle Extraktion einzelner Embryonen aus Samen mit feinen Werkzeugen 5. Allerdings ist LCM Ausrüstung nicht allgemein verfügbar und manuelle Extraktion Embryos in frühen Stadien ist zeitaufwendig und erfordern hervorragende Präparation Fähigkeiten, die nicht leicht übertragbar. Neben genomweite Analysen, in situ Genexpressionsanalysen sind auch schwierig, auf junge, whole-mount Embryonen von Arabidopsis durchzuführen. In gewissem Umfang können junge Embryonen auf Objektträgern durch sanften Druck auf die Samen freigesetzt werden und für Reportergen-Assays oder Protein Erkennung durch Immunfärbung (siehe zum Beispiel 6,7). Diese Technik ist jedoch nicht erlaubt High-Throughput-Embryo Isolierung und behindern so quantitative Analysen. Deshalb entwickelten wir eine effiziente und schnelle Protokollfür frühe Embryo isoliert von Arabidopsis Samen, die einfach zu installieren, leicht übertragbar und eignet sich für eine Vielzahl von nachgelagerten Anwendungen. Das Grundprinzip besteht darin, vorsichtig zerdrücken Samen - seziert von jungen Schoten in ein Eppendorf-Röhrchen mit einem Kunststoff-Stößel in einer geeigneten Isolierung Puffer. Der Extrakt wird in Tröpfchen auf einer Multi-Well-Objektträger aufgebracht und auf das Vorhandensein des freigesetzten Embryonen bei der gewünschten Stufe gescreent unter Verwendung eines invertierten Mikroskops. Die Embryonen werden gesammelt mit einem Glas zu einem Mikroinjektor oder einem Standard-Labor-Pipette angebracht mikrokapillaren. Für molekulare Anwendungen werden Embryonen zweimal durch wiederholte Freisetzung in Tropfen neue Isolierung Puffer vor der Übertragung auf den Zielpuffer in einem minimalen Volumen gewaschen. Für zytologische Anwendungen (Reporter-Assays, Immunfärbung FISH), können Waschschritte verzichtet werden. Das Verfahren bietet mehrere Vorteile: (i) es ergibt 25-40 Embryonen in ~ 45 min für die zytologische Appfentlichungen oder in 3-4 Stunden für die molekulare Anwendungen (einschließlich der Waschschritte), (ii) es ermöglicht Isolierung spezifischer embryonalen Stadien, (iii) es ist leicht auf andere Personen übertragbar und Labors aufgrund seiner einfachen Setup, (iv) es erfordert erschwinglichen Geräten für die Grundeinstellungen, die zugänglich für Upgrades ist, und (v) wurde erfolgreich für verschiedene nachgelagerte Anwendungen wie RNA-Sequenzierung 8, Gen-spezifischen DNA-Methylierung Analyse 9, Reporter-Assays (10 und Raissig et al. verwendet, in prep.) und FISH (J. Jaenisch, U. Grossniklaus, C. Baroux unveröffentlicht, siehe Abbildung 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

In Blütenpflanzen, entwickelt der Embryo innerhalb einer nährenden Gewebe - das Endosperm - von den mütterlichen Saatgut Integumente (oder Samenschale) umgeben ist. Als Folge wird die Isolierung von pflanzlichen Embryonen in frühen Stadien (1 Zelle Kugelsternhaufen Stufe) technisch anspruchsvolle aufgrund ihrer relativen Unzugänglichkeit. Effiziente manuelle Zerlegung in frühen Stadien ist stark durch die geringe Größe der jungen Arabidopsis Samen und die Haftfähigkeit des Embryos in die umliegenden Gewebe beeinträchtigt. Hier beschreiben wir eine Methode, die die effiziente Isolierung von jungen Arabidopsis Embryonen ermöglicht, wodurch bis zu 40 Embryonen in 1 Stunde bis 4 Stunden, abhängig von der nachgeschalteten Anwendung. Die Embryonen werden in die Isolation Puffer durch leichtes Quetschen 250-750 Samen mit einer Kunststoff-Stößel in ein Eppendorf-Röhrchen veröffentlicht. Ein Glas entweder mit einem Standard-Labor-Pipette angebracht mikrokapillaren (über einen Gummischlauch) oder eine hydraulisch gesteuerte Mikroinjektor wird verwendet, um Embryonen aus Tröpfchen Ort zu sammelnd auf einer Multi-Well-Folie auf einem invertierten Lichtmikroskops. Die technischen Fähigkeiten erforderlich sind einfach und leicht übertragbar, und die grundlegende Einrichtung erfordert keine teure Ausrüstung. Erhaltene Embryonen sind geeignet für eine Vielzahl von Downstream-Anwendungen wie RT-PCR, RNA-Sequenzierung, DNA-Methylierungsanalyse, Fluoreszenz-in situ-Hybridisierung (FISH), Immunfärbung und Reportergen-Assays.

Unsere Embryo Isolation Verfahren (Abbildung 1) ermöglicht die Isolierung von bis zu 40 Embryonen in 4 Stunden, wenn Waschungen durchgeführt werden, z. B. für die molekulare Anwendungen oder in weniger als einer Stunde, wenn Wäschen weggelassen werden, zB für die zytologische Anwendungen. Abbildung 2 zeigt hohe und niedrige Qualität mikrokapillaren Tipps und Abbildung 3 zeigt den Aufbau des Embryos Isolierung Maschine. Abbildung 4</strong...

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

Wir berichten über eine effiziente und einfache Methode, um Embryonen in frühen Stadien der Entwicklung Isolat aus<em&gt; Arabidopsis thaliana</em&gt; Samen. Bis zu 40 Embryonen in 1 Stunde bis 4 Stunden isoliert werden, abhängig von der Downstream-Anwendungen. Das Verfahren eignet sich für die Transkriptom-, DNA-Methylierung, die Expression des Reportergens, Immunfärbung und Fluoreszenz<em&gt; In situ</em&gt; Hybridisierung analysiert.

Effiziente und schnelle Isolierung von Frühphasen-Embryonen aus<em&gt; Arabidopsis thaliana</em&gt; Seeds

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

efficient-rapid-isolation-early-stage-embryos-from-arabidopsis

Research

JoVE Journal

Biology

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

17.7K Aufrufe.  University of Zürich. Wir berichten über eine effiziente und einfache Methode, um Embryonen in frühen Stadien der Entwicklung Isolat aus Arabidopsis thaliana Samen. Bis zu 40 Embryonen in 1 Stunde bis 4 Stunden isoliert werden, abhängig von der Downstream-Anwendungen. Das Verfahren eignet sich für die Transkriptom-, DNA-Methylierung, die Expression des Reportergens, Immunfärbung und Fluoreszenz In situ Hybridisierung analysiert.

Serie: 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.

Platzieren Growth Factor-Coated Perlen auf Early Stage Hühnerembryonen

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

Forschung

Biologie

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.

Isolation von Protoplasten aus Geweben von 14-Tage alte Keimlinge von 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 Transformation von Arabidopsis thaliana Zum Untersuchen PTSO2: β-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 ...

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 Polar Glycerolipid Profiling durch Dünnschichtchromatographie (TLC) mit Gas-Flüssigkeits-Chromatographie (GLC) Coupled

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.

Chromatin Immunopräzipitation-Assay für Tissue-spezifische Gene mit Frühzeitige muriner Embryonen

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.

Isolierung und Reinigung von Kinesin aus Drosophila Embryonen

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.

Schnelle und effiziente Erzeugung von Neuronen aus menschlichen pluripotenten Stammzellen in einem Multititre Platte Format

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.

Metabolische Markierung und Membran Fraktionierung für Vergleichende Proteomanalyse von<em&gt; Arabidopsis thaliana</em&gt; Fahrwerk Zellkulturen

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.

Histochemische Färbung von<em&gt; Arabidopsis thaliana</em&gt; Sekundärzellwand-Elemente

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.

Standardisierte Methode für Hochdurchsatz-Sterilisation von Arabidopsis Samen

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