The authors thank Davy Opdenacker for technical assistance and photography. We greatly thank Dr. Annick Bleys for helpful suggestions to improve the manuscript. This work was financed by the Interuniversity Attraction Poles Programme IUAP P7/29 'MARS' from the Belgian Federal Science Policy Office, by the FWO grant G027313N and by the Agency for Innovation by Science and Technology, IWT (IR).

1. Arabidopsis Lris protocollo Nota: Il testo fa riferimento a &quot;piccoli&quot; o &quot;grandi&quot; esperimenti scala. Esperimenti su scala ridotta, come l&#39;analisi linea marcatore e colorazione istologica 6, 14, richiedono solo un paio di esempi. Esperimenti su vasta scala, come quantitativa real-time qRT-PCR, microarray 9-11 o RNA sequencing, richiedono una maggiore quantità di campioni. In quanto tale, un importo pari a ~ 1000 piantine per campione è stato utilizzato da Vanneste et al. 11 per eseguire esperimenti di microarray dopo segmento radice dissezione. GIORNO 1 <ol><li> Sterilizzazione di Arabidopsis Seeds Nota: selezionare una delle seguenti procedure per la sterilizzazione del seme. Uno di essi è adatto per le prossime fasi del protocollo. Sterilizzazione a gas (1.1.2) presenta due vantaggi principali rispetto sterilizzazione liquida (1.1.1): ci vuole meno tempo quando si maneggia una grande intorpiditoer di semi, poiché nessuna pipettaggio deve verificarsi per i singoli campioni; ei semi sterilizzati possono essere conservati per un periodo più lungo. Tuttavia, esso richiede un essiccatore e accurato trattamento. <ol><li> Sterilizzazione liquido <ol><li> Versare il numero desiderato di semi in tubi di micro-centrifuga. Utilizzare fino a 20 mg (~ 800 semi) in una provetta da 1,5 ml o 40 mg (~ 1600 semi) in 2 ml provette micro-centrifuga. </li><li> Aggiungere 1 ml di etanolo al 70%. Invertire o scuotere delicatamente per 2 minuti. </li><li> Sostituire l&#39;etanolo al 70% con 1 ml di soluzione di sterilizzazione per 15 min. (Preparare la soluzione di sterilizzazione fresco: 3,85 ml di ipoclorito di sodio (NaOCl) stock (12%) (ATTENZIONE: l&#39;utilizzo fumi cappa), 5 ml Tween 20, fino a 10 ml con acqua invertire i tubi diverse volte per assicurarsi che tutti i semi arrivano. in contatto con la soluzione. </li><li> In una cappa a flusso laminare, sostituire la soluzione di sterilizzazione con 1 ml di acqua distillata sterile e sciacquare i semi 5 volte per 5 min da ripetutily sostituzione con 1 ml di acqua distillata sterile fresca. </li></ol></li><li> Sterilizzazione Gas <ol><li> Versare il numero desiderato di semi in un tubo da centrifuga micro-2 ml. Utilizzare singoli tubi quando si lavora con più linee indipendenti, e disporli aperti in una scatola tubi micro-centrifuga di plastica o di supporto. Se il numero di semi in un tubo supera 500 (12,5 mg), suddividere i semi al numero appropriato di provette per assicurare sterilizzazione. Assicurarsi che i tappi di tubi adiacenti non coprono l&#39;un l&#39;altro. Montare insieme il coperchio della scatola sul fondo, in quanto deve essere nell&#39;essiccatore anche per la sterilizzazione. </li><li> Posizionare la scatola nella ciotola di un essiccatore, insieme a un bicchiere di vetro (ATTENZIONE: l&#39;utilizzo dei fumi cappa). </li><li> Riempire il bicchiere con 100 ml di 12% NaOCl (ATTENZIONE: Utilizzare cappa). Mettere il coperchio sulla essiccatore, ma lasciare una piccola apertura in cui è collocato il bicchiere. </li><li> Aggiungere 3 ml di acido cloridrico 37% (fumigante) (CAUZIONE: Usa fume hood) al bicchiere attraverso la piccola apertura e chiudere rapidamente un essiccatore. La volontà bolla soluzione per qualche tempo a causa di Cl formazione 2 -gas. Lasciare il sistema chiuso O / N (o almeno 8 ore). </li><li> Togliere il coperchio del essiccatore. Estrarre la scatola e coprire con il coperchio per evitare la contaminazione durante il trasporto. </li><li> Mettere la scatola in un armadio flusso laminare, togliere il coperchio e lasciare i semi per circa 1 ora a temperatura ambiente per permettere la fuoriuscita di gas. Nota: I semi possono essere secca in tubi di micro-centrifuga chiusi conservati in modo sicuro fino a 2 settimane a 4 ° C. </li></ol></li></ol></li><li> Laterale induzione Root in Arabidopsis <ol><li> Laterale Root inibizione (NPA Treatment) <ol><li> Preparare il terreno di coltura per la crescita in vitro. Il terreno contiene metà forza Murashige e Skoog miscela di sale 15, 0,1 g / L mio-inositolo, 10 g / L di saccarosio, ed è tamponata con 0,5g / L 2- (N-morfolino) Acido etansolfonico (MES). </li><li> Regolare il pH a 5,7 con 1 M KOH. Aggiungere 8 g / L di agar pianta tessuto e il mezzo autoclave per 20 minuti a 121 ° C. </li><li> Preparare una soluzione madre 25 mm di NPA in dimetilsolfossido (DMSO) (ATTENZIONE: Indossare guanti). In una cappa a flusso laminare, raffreddare il mezzo autoclavato fino a circa 65 ° C (che è la temperatura a cui la bottiglia può essere tenuto a mano), e aggiungere 25 soluzione madre NPA mM per ottenere una concentrazione finale di 10 pM NPA. </li><li> Omogeneizzare su oltre agitando delicatamente il flacone per 10 secondi. Versare 50 ml di terreno per ogni piatto quadrato di Petri (12 cm x 12 cm). Nota: In caso di esperimenti su larga scala, applicare una rete di nylon (20 micron) per assicurare un facile trasferimento. Preparare una maglia (9 cm x 9 cm) per la sterilizzazione in autoclave (Figura 1A). Quando autoclave, applicare la rete al mezzo di crescita con una pinzetta sterile (Figura 1B). Spingere delicatamente la maglia al mezzo di crescita con una sterilized drigalsky per assicurarsi che sia in buon contatto con il mezzo di crescita (Figura 1B) (ATTENZIONE: Il lavoro in condizioni sterili). </li><li> Seminare i semi sui NPA piastre. Nota: Nel caso in cui è previsto un esperimento su larga scala, seminare 2 file densi e ben allineati di 50 semi di facilitare il prelievo (vedi 1.2.2.3). <ol><li> In caso di sterilizzazione liquido, seminare i semi su NPA-lastre con una pipetta 200 l. Tagliare 3 - 4 mm dalla fine delle punte di pipettaggio in condizioni sterili (o prima della sterilizzazione delle punte) per poter pipettare i semi. Pipettare su e giù un paio di volte a ri-sospendere i semi, poi pipettare acqua circa 150 microlitri sterile con 10 - 20 semi e aspettare che il sedimento semi sul fondo della punta. (ATTENZIONE: Il lavoro in condizioni sterili) Nota: Toccando l&#39;agar o mesh delicatamente con la punta, una goccia d&#39;acqua con un seme sarà rilasciato da esso (Figura 1C). </li> &lt;li&gt; In caso di sterilizzazione a gas, seminare i semi con autoclave toothpicks.Pinch la stuzzicadenti nel agar prima di prendere i semi per garantire una superficie adesiva. Raccogliete la semi uno a uno con lo stuzzicadenti e distribuire i semi sulle piastre (Figura 1D). In alternativa, aggiungere acqua sterile per i semi, e di seminare come descritto in 1.2.1.5.1 (ATTENZIONE: Il lavoro in condizioni sterili). </li></ol></li><li> Sigillare le piastre con nastro adesivo traspirante e memorizzarli piastre a 4 ° C al buio per almeno 2 giorni e massimo una settimana. Questo è necessario per stratificazione e garantisce sincronizzati e germinazione più efficiente. Nota: In alternativa, i semi possono essere stratificati prima della semina, immerse in acqua distillata in tubi micro-centrifuga, che richiede meno spazio frigorifero. In questo caso, conservare le provette per almeno una settimana a 4 ° C, e spostare le piastre al mobile crescita subito dopo la semina (vedi 1.2.1.7). GIORNO 3 </li&gt; <li> Spostare le piastre al mobile crescita (luce continua (110 μE m -2 s -1), 21 ° C) per 5 giorni (2 giorni di germinazione e 3 giorni di crescita) in posizione quasi verticale (80 a 90 °) per assicurare la crescita delle radici, e non nel mezzo. GIORNO 8 </li></ol></li><li> Laterale Root induzione (NAA Treatment) <ol><li> Preparare lo stesso terreno di coltura come descritto in 1.2.1.1 e 1.2.1.2. Preparare una soluzione 50 mM magazzino di NAA in DMSO (ATTENZIONE: Indossare guanti). Raffreddare il terreno autoclavato fino a 65 ° C e aggiungere la soluzione NAA al mezzo per ottenere una concentrazione finale di 10 mM NAA (NOTA: Il lavoro in condizioni sterili). <ol><li> Omogeneizzare su oltre agitando delicatamente il flacone per 10 secondi. Versare 50 ml di terreno per ogni piatto quadrato di Petri (12 cm x 12 cm). </li></ol></li><li> Trasferire le piantine dalle piastre contenenti terreno di coltura supplementato con 10 micron NPA a quelli supsere realizzata con 10 micron NAA. <ol><li> Agganciare le braccia di pinzette ricurve sotto i cotiledoni della piantina e rimuoverla delicatamente dalla piastra. Trasferire solo piantine le cui radici sono cresciuti completamente in contatto con il mezzo NPA-crescita e preferibilmente verso il basso. </li><li> Skim la radice nel medio di crescita della superficie-NAA che contiene oltre una piccola distanza. Assicurarsi che le radici delle piante sono in buon contatto con il mezzo di crescita. Se necessario, premere il radice delicatamente sulla superficie terreno di coltura con le pinzette. Nota: Nel caso di un grande esperimento, rimuovere tutti piantine che non sono in contatto con la rete e trasferimento sollevando la mesh ai due angoli superiori con pinzette. Assicurarsi che la maglia è in buon contatto con il mezzo-NAA contenente schiumando maglia sulla superficie agar su una piccola distanza (Figura 1E). </li></ol></li><li> Sigillare le nuove piastre con nastro adesivo traspirante e metterli nel quadro di crescita (conluce continuo (110 μE m -2 s -1), 21 ° C) in posizione verticale per il tempo desiderato dopo induzione fino al campionamento. Nota: Nel caso di un grande esperimento, utilizzare un bisturi per tagliare i segmenti radice tutti in una volta direttamente sulla maglia e campionare raschiando delicatamente la superficie della mesh. </li></ol></li></ol></li></ol> 2. Lris mais protocollo <ol><li> Stratificazione di mais kernel <ol><li> Incubare i chicchi di mais a 4 ° C al buio per almeno 1 settimana. Nota: Questo protocollo è stato sviluppato utilizzando la linea inbred di mais B73. </li></ol></li><li> Sterilizzazione di mais kernel GIORNO 1 Nota: Anche se piante di mais non devono essere coltivate in condizioni di sterilità, si consiglia di sterilizzare i chicchi di mais e di lavorare con materiale sterile per prevenire la crescita di funghi nel rotolo di cartasistema. <ol><li> Mettere il numero necessario di chicchi di mais in un bicchiere di vetro sterilizzati. </li><li> Aggiungere 100 ml di soluzione di sterilizzazione (6% NaOCl in acqua) (ATTENZIONE: l&#39;utilizzo cappa). </li><li> Aggiungi un ancoretta magnetica e posizionare il bicchiere su un agitatore magnetico a bassa velocità di agitazione (circa 250 giri al minuto) a temperatura ambiente per 5 minuti. </li><li> Risciacquare cinque volte per 5 min, sostituendo la soluzione con 100 ml di acqua fresca sterile. </li></ol></li><li> La semina mais kernel <ol><li> Indossare guanti per ridurre il rischio di contaminazione, e prendere un rotolo di carta assorbente. </li><li> Strappare due tratti di mano un tovagliolo di carta con una lunghezza totale di due fogli (circa 92 centimetri x 24 cm) e metterli uno sopra l&#39;altro su una superficie pulita (Figura 2A). </li><li> Piegare i fogli raddoppiare la lunghezza (circa 92 cm x 12 cm) (Figura 2A). </li><li> Utilizzare le pinzette per distribuire 10 kernel a circa 2 cm dalla parte superiore su tegli intera lunghezza della carta, mantenendo un&#39;intercapedine di 8 cm tra ogni kernel e 8 cm connessione alle due estremità (Figura 2B). Assicurarsi che la radichetta del kernel sia rivolta verso il basso e verso la carta (Figura 2B). </li><li> Rotolare delicatamente la carta su tutta la lunghezza, pur mantenendo i semi in sede (Figura 2B). Per facilitare il rotolamento, è facoltativo spruzzare prima la carta con acqua sterile. </li><li> Mettere i rotoli di carta in tubi di vetro sterilizzati di circa 6 cm di diametro e 14 cm di altezza (per esempio, 250 ml provette centrifuga) e metterli in un rack (Figura 2C). </li></ol></li><li> Laterale induzione Root in mais <ol><li> Preparare una soluzione al 50 NPA mM magazzino (disciolto in DMSO) (ATTENZIONE: Indossare guanti). </li><li> Per ogni tubo, fare una soluzione al 50 micron NPA aggiungendo 125 microlitri della soluzione madre 50 NPA mM a 125 ml di acqua sterile, in un bicchiere di vetro sterilizzati. </li><li> Mescolare bene eversare 125 ml di soluzione 50 mM NPA sopra il rotolo di carta nel tubo. Il rotolo di carta in grado di assorbire la soluzione e diventare completamente assorbito. Nota: Quando si utilizzano rotoli di carta e tubi con altre dimensioni, regolare il volume di conseguenza. Il liquido dovrebbe raggiungere a metà strada del tubo per assicurare l&#39;assorbimento sufficiente. </li><li> Collocare il sistema rotolo di carta in un armadio di crescita per tre giorni (27 ° C, luce continua, il 70% di umidità relativa). Assicurarsi che i rotoli di carta rimangono impregnati con l&#39;aggiunta extra di 50 micron soluzione NPA, dal momento che la soluzione evapora nel tempo (Figura 2D). GIORNO 4 </li><li> Preparare una soluzione al 50 NAA mM magazzino (disciolto in DMSO) (ATTENZIONE: Indossare guanti). </li><li> Per ogni tubo, preparare una soluzione NAA 50 micron con l&#39;aggiunta di 125 microlitri dal magazzino 50 NAA mM a 125 ml di acqua sterile in un bicchiere di vetro sterilizzati. </li><li> Premere delicatamente la maggior parte della rimanente soluzione di NPA dai rotoli di carta e posizionare ilm di acqua sterile per 5 minuti per lavare fuori (ATTENZIONE: Indossare guanti). Spremere delicatamente la maggior parte dell&#39;acqua dai rotoli di carta. Ripetere questa operazione di lavaggio per tre volte. </li><li> Mescolare bene e versare la soluzione NAA 50 micron sui rotoli di carta nei tubi. Assicurarsi che siano completamente immersi. </li><li> Posizionare il sistema rotolo di carta nel gabinetto crescita (27 ° C, luce continua, 70% di umidità relativa) per la durata desiderata dopo induzione. </li></ol></li><li> Laterale induzione Root in accidentale Corona Roots of Maize Nota: Il Lris come descritto sopra induce radici laterali nella radice primaria. Tuttavia, nel mais e altre monocotiledoni, la radice primaria e le radici seminali embrionali, insieme con le loro radici laterali, sono principalmente importante durante lo sviluppo piantina. In una fase successiva della crescita delle piante, le radici avventizie postembrionali sorgono sullo stelo e anche sviluppare radici laterali per formare un nuovo sistema di radice 16. Il Lris può anche essere utilizzatod per indurre radici laterali su queste post-embrionali radici avventizie corona seguendo alcuni piccoli aggiustamenti alla Lris sulla radice primaria embrionale. <ol><li> Per rendere i rotoli di carta, creare un sandwich di carte con rispettivamente due strati di mano tovagliolo di carta sul lato esterno, e due strati di carta all&#39;interno di germinazione. Posizionare i kernel sterilizzati (vedere i passi 2,1-2,2) tra i due fogli di carta germinazione. Carta germinazione, rispetto alla carta asciugamani, sarà meno incline ad essere penetrato da peli radicali e radici laterali, che renderà l&#39;apertura del rotoli di carta più facile in seguito. D&#39;altra parte, i due strati esterni di asciugamano di carta saranno facilitare l&#39;assorbimento del liquido. </li><li> Inserire i rulli in 700 ml di tubi di vetro e versare acqua sterile senza NPA su di loro. Assicurarsi che siano completamente immersi. </li><li> Germinare i chicchi sterilizzati nell&#39;armadio di crescita (vedi punto 2.4.9). </li><li> Crescere le piante fino alle radici avventizie coronainiziare ad emergere (6 giorni per B73). Assicurarsi che i rotoli di carta sono tenute umido in ogni momento con l&#39;aggiunta di acqua sterile quando necessario. </li><li> Trasferimento a una soluzione 25 mM NPA per 4 giorni (vedi i punti 2.4.1 a 2.4.4), seguita da una simile induzione di apertura laterale radice sostituendo la soluzione con una soluzione NPA NAA 50 pM dopo una fase di lavaggio (vedi passo 2.4 0,5 a 2.4.9). </li></ol></li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-capture+microdissection,+a+tool+for+the+global+analysis+of+gene+expression+in+specific+plant+cell+types:+identification+of+genes+expressed+differentially+in+epidermal+cells+or+vascular+tissues+of+maize.">Laser-capture microdissection, a tool for the global analysis of gene expression in specific plant cell types: identification of genes expressed differentially in epidermal cells or vascular tissues of maize.</a> Plant Cell. 15, 583-596 (2003).</li><li>Ludwig, Y., Hochholdinger, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+microdissection+of+plant+cells.">Laser microdissection of plant cells.</a> Methods Mol Biol. 1080, 249-258 (2014).</li><li>Beeckman, T., Engler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+easy+technique+for+the+clearing+of+histochemically+stained+plant+tissue.">An easy technique for the clearing of histochemically stained plant tissue.</a> Plant Mol. Biol. Rep. 12, 37-42 (1994).</li><li>Ditengou, F. A., 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=Mechanical+induction+of+lateral+root+initiation+in+Arabidopsis+thaliana.">Mechanical induction of lateral root initiation in Arabidopsis thaliana.</a> Proc. Natl. Acad. Sci. USA. 105, 18818-18823 (2008).</li><li>Lucas, M., Godin, C., Jay-Allemand, C., Laplaze, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auxin+fluxes+in+the+root+apex+co-regulate+gravitropism+and+lateral+root+initiation.">Auxin fluxes in the root apex co-regulate gravitropism and lateral root initiation.</a> J. Exp. Bot. 59, 55-66 (2008).</li><li>Wang, S., Taketa, S., Ichii, M., Xu, L., Xia, K., Zhou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+root+formation+in+rice+(Oryza+Sativa.+L.):+differential+effects+of+indole-3-acetic+acid+and+indole-3-butyric+acid.">Lateral root formation in rice (Oryza Sativa. L.): differential effects of indole-3-acetic acid and indole-3-butyric acid.</a> Plant Growth Regul. 41, 41-47 (2003).</li><li>Martinez-de la Cruz, E., Garcia-Ramirez, E., Vazquez-Ramos, J. M., Reyes de la Cruz, H., Lopez-Bucio, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auxins+differentially+regulate+root+system+architecture+and+cell+cycle+protein+levels+in+maize+seedlings.">Auxins differentially regulate root system architecture and cell cycle protein levels in maize seedlings.</a> J Plant Physiol. 176, 147-156 (2015).</li><li>Beyer, E. M., Morgan, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ethylene+on+uptake,+distribution,+and+metabolism+of+indoleacetic+acid-1-14C+and+-2-14C+and+naphthaleneacetic+acid-1-14C.">Effect of ethylene on uptake, distribution, and metabolism of indoleacetic acid-1-14C and -2-14C and naphthaleneacetic acid-1-14C.</a> Plant Physiol. 46, 157-162 (1970).</li><li>Delbarre, A., Muller, P., Imhoff, V., Guern, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+mechanisms+controlling+uptake+and+accumulation+of+2,4-dichlorophenoxy+acetic+acid,+naphthalene-1-acetic+acid,+and+indole-3-acetic+acid+in+suspension-cultured+tobacco+cells.">Comparison of mechanisms controlling uptake and accumulation of 2,4-dichlorophenoxy acetic acid, naphthalene-1-acetic acid, and indole-3-acetic acid in suspension-cultured tobacco cells.</a> Planta. 198, 532-541 (1996).</li><li>Bailey-Serres, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microgenomics:+genome-scale,+cell-specific+monitoring+of+multiple+gene+regulation+tiers.">Microgenomics: genome-scale, cell-specific monitoring of multiple gene regulation tiers.</a> Annu Rev Plant Biol. 64, 293-325 (2013).</li><li>Perez-Torres, C. A., 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=Phosphate+availability+alters+lateral+root+development+in+Arabidopsis+by+modulating+auxin+sensitivity+via+a+mechanism+involving+the+TIR1+auxin+receptor.">Phosphate availability alters lateral root development in Arabidopsis by modulating auxin sensitivity via a mechanism involving the TIR1 auxin receptor.</a> Plant Cell. 20, 3258-3272 (2008).</li></ol>

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Nel protocollo Arabidopsis Lris, è importante trasferire solo le piantine che sono cresciuti completamente in contatto con il terreno di coltura contenente NPA. Questo assicura che laterale iniziazione radice è bloccato su tutta la lunghezza della radice. Al fine di prevenire il ferimento di piantine durante il trasferimento, le braccia delle pinze curve possono essere agganciati sotto i cotiledoni della piantina. Al trasferimento, assicurarsi che le radici di piantine sono in contatto sufficiente con il terr...

Il sistema radicale è cruciale per la crescita delle piante, in quanto garantisce l&#39;ancoraggio e l&#39;assorbimento di acqua e nutrienti dal terreno. Poiché l&#39;espansione di un sistema radicale si basa principalmente sulla produzione di radici laterali, la loro iniziazione e formazione sono stati ampiamente studiati. Radici laterali sono iniziati in uno specifico sottogruppo di cellule pericycle, chiamate cellule fondatore 1. Nella maggior parte delle dicotiledoni, come Arabidopsis thaliana, queste cellule si trovano ai poli protoxylem 2, mentre in monocotiledoni, come il mais, si trovano ai poli floema 3. Fondatore cellule sono caratterizzati da un aumento della risposta auxina 4, seguita dalla espressione di specifici geni del ciclo cellulare (ad esempio, CYCLIN B1; 1 / CYCB1; 1), dopo di che subiscono un primo giro di asimmetrici divisioni anticlinali 5. Dopo una serie di divisioni anticlinali e periclinal coordinati, un primordio radice laterale è formata che finalmente emergerà come unradici laterali utonomous. La posizione e la tempistica di apertura laterale root non sono comunque prevedibile, poiché questi eventi sono né abbondanti né sincronizzato. Questo impedisce l&#39;utilizzo di approcci molecolari, come trascrittomica per studiare questo processo. Per affrontare questo, una laterale Root inducibile System (Lris) è stato sviluppato 6, 7. In questo sistema, piantine vengono prima trattati con acido N -1-naphthylphthalamic (NPA), che inibisce il trasporto e l&#39;accumulo di auxina, bloccando di conseguenza laterali iniziazione radice 8. Con successiva cessione delle piantina di terreno contenente l&#39;auxina sintetico acido acetico 1-naftalene (NAA), l&#39;intero strato pericycle risponde ai livelli di auxina elevati così massicciamente che inducono alla radice l&#39;avvio divisioni cellulari laterali 6. Come tale, questo sistema porta a veloci, sincroni ed estese radici laterali iniziazioni, che consente un facile prelievo di campioni di radice arricchito da una determinata fase di ritardolo sviluppo delle radici ral. Successivamente, questi campioni possono essere utilizzati per determinare profili di espressione a livello di genoma durante la formazione delle radici laterali. Il Lris ha prodotto conoscenze già significativo circa iniziazione laterale radici in Arabidopsis e mais 9-13, ma la necessità di applicare questo sistema per altre specie di piante diventa più evidente quanto più genomi sono in sequenza e non vi è un interesse crescente per trasferire la conoscenza a economico importante specie. Qui, sono indicati i protocolli dettagliati della Arabidopsis e mais LRISs. Successivamente, un esempio di utilizzo del sistema è fornito, illustrando come trascrittomica dati ricavati dal mais Lris possono essere utilizzati per identificare omologhi funzionali che hanno una funzione conservata durante l&#39;inizio radice laterale attraverso diverse specie vegetali. Infine, le linee guida per ottimizzare il Lris sono proposti per altre specie vegetali.

<table border="0" cellpadding="0" cellspacing="0" width="1258">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">ARABIDOPSIS LRIS</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td style="width:551px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Seeds</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Arabidopsis seeds</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td>Col-0 ecotype</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Gas sterilization of seeds</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">micro-centrifuge tubes 1.5 ml</td>
			<td style="width:228px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">0030 125.215</td>
			<td>Eppendorf microtubes 3810X, PCR clean</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">micro-centrifuge tubes 2 ml</td>
			<td style="width:228px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">0030 120.094</td>
			<td>Eppendorf Safe-Lock microcentrifuge tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">hydrochloric acid</td>
			<td style="width:228px;">Merck KGaA</td>
			<td style="width:119px;">1,003,171,000</td>
			<td>37% (fuming) for analysis EMSURE ACS,ISO,Reag. Ph Eu</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">glass desiccator</td>
			<td style="width:228px;">SIGMA-ALDRICH</td>
			<td style="width:119px;"></td>
			<td>Pyrex</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:361px;">glass beaker</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">plastic micro-centrifuge tubes box or holder</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Bleach sterilization of seeds</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">ethanol</td>
			<td style="width:228px;">Chem-Lab nv</td>
			<td style="width:119px;">CL00.0505.1000</td>
			<td>Ethanol, abs. 100% a.r. dilute to 70%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">sodium hypochlorite (NaOCl)</td>
			<td style="width:228px;">Carl Roth</td>
			<td style="width:119px;">9062.3</td>
			<td>12%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Tween 20</td>
			<td style="width:228px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">P1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">sterile water</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Growth medium</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Murashige and Skoog salt mixture</td>
			<td style="width:228px;">DUCHEFA Biochemie B.V.</td>
			<td style="width:119px;">M0221-0050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">myo-inositol</td>
			<td style="width:228px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">I5125-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">2-(N-morpholino)ethanesulfonic acid (MES)</td>
			<td style="width:228px;">DUCHEFA Biochemie B.V.</td>
			<td style="width:119px;">M1503.0100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">sucrose</td>
			<td style="width:228px;">VWR, Internation LLC</td>
			<td style="width:119px;">27483.294</td>
			<td>D(+)-Sucrose Ph. Eur.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">KOH</td>
			<td style="width:228px;">Merck KGaA</td>
			<td style="width:119px;">1050211000</td>
			<td>pellets for analysis (max. 0.002% Na) EMSURE ACS,ISO,Reag. Ph Eur</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Plant Tissue Culture Agar</td>
			<td style="width:228px;">LabM Limited</td>
			<td style="width:119px;">MC029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Lateral root induction chemicals</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">N-1-naphthylphthalamic acid (NPA)</td>
			<td style="width:228px;">DUCHEFA Biochemie B.V.</td>
			<td style="width:119px;">No. N0926.0250</td>
			<td>10 &#181;M (Arabidopsis)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">1-naphthalene acetic acid (NAA)</td>
			<td style="width:228px;">DUCHEFA Biochemie B.V.</td>
			<td style="width:119px;">No. N0903.0050</td>
			<td>10 &#181;M (Arabidopsis)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">dimethylsulfoxide (DMSO)</td>
			<td style="width:228px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">494429-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Making a mesh for transfer</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">nylon mesh</td>
			<td style="width:228px;">Prosep byba</td>
			<td style="width:119px;"></td>
			<td>Synthetic nylon mesh 20 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Sowing and seedling handling</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">square petri dish plates</td>
			<td style="width:228px;">GOSSELIN</td>
			<td style="width:119px;">BP124-05</td>
			<td>12 x 12 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">50 ml DURAN tubes</td>
			<td style="width:228px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">CLS430304</td>
			<td>Corning&#160;50 mL centrifuge tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">drigalski</td>
			<td style="width:228px;">Carl Roth</td>
			<td style="width:119px;">K732.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">pipette</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">cut pipette tips</td>
			<td style="width:228px;">Daslab</td>
			<td style="width:119px;">162001X</td>
			<td>Universal 200, cut off 5 mm of tip before autoclaving</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">breathable tape&#160;</td>
			<td style="width:228px;">3M Deutschland GmbH</td>
			<td style="width:119px;">cat. no. 1530-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">tweezers</td>
			<td style="width:228px;">Fiers nv/sa</td>
			<td style="width:119px;">K342.1; K344.1</td>
			<td>Dumont tweezers type a nr 5; Dumont tweezers type e nr 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Growth conditions</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">growth room</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td>21 &#176;C, continuous light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Materials</td>
			<td style="width:228px;">Company</td>
			<td style="width:119px;">Catalog</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">MAIZE LRIS</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Seeds</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Maize kernels</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td>B-73</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Bleach sterilization of kernels</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:361px;">glass beaker</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">magnetic stirrer&#160;</td>
			<td style="width:228px;">Fiers nv/sa</td>
			<td style="width:119px;">C267.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">sodium hypochlorite (NaOCl)</td>
			<td style="width:228px;">Carl Roth</td>
			<td style="width:119px;">9062.3</td>
			<td>12%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">sterile water</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Lateral root induction chemicals</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">N-1-naphthylphthalamic acid (NPA)</td>
			<td style="width:228px;">DUCHEFA Biochemie B.V.</td>
			<td style="width:119px;">No. N0926.0250</td>
			<td>50 &#181;M (maize primary root), 25 &#181;M (maize adventitious root)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">1-naphthalene acetic acid (NAA)</td>
			<td style="width:228px;">DUCHEFA Biochemie B.V.</td>
			<td style="width:119px;">No. N0903.0050</td>
			<td>50 &#181;M (maize)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">dimethylsulfoxide (DMSO)</td>
			<td style="width:228px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">494429-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Sowing and seedling handling</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">paper hand towels</td>
			<td style="width:228px;">Kimberly-Clark Professional*</td>
			<td style="width:119px;">6681</td>
			<td>SCOTT Hand Towels - Roll / White; sheet size (24 x 46 cm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">seed germination paper</td>
			<td style="width:228px;">Anchor Paper Company</td>
			<td style="width:119px;"></td>
			<td>10 X 15 38# seed germination paper</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">tweezers</td>
			<td style="width:228px;">Fiers nv/sa</td>
			<td style="width:119px;">K342.1; K344.1</td>
			<td>Dumont tweezers type a nr 5; Dumont tweezers type e nr 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">250 ml (centrifuge) tubes</td>
			<td style="width:228px;">SCHOTT DURAN</td>
			<td style="width:119px;">2160136</td>
			<td>approx. 5.6 cm diameter and 14.7 cm height&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">700 ml tubes</td>
			<td style="width:228px;">DURAN GROUP</td>
			<td style="width:119px;">213994609</td>
			<td>cylinders, round foot tube, D 60&#160; x 250</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rack</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td>for maize tubes, home made</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">sterile water</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Growth conditions</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">growth cabinet</td>
			<td style="width:228px;"></td>
			<td style="width:119px;"></td>
			<td>27 &#176;C, continuous light, 70% relative humidity</td>
		</tr>
	</tbody>
</table>

lateral root inducible system in arabidopsis and maize

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

Applicazione del Lris per eseguire Trascrittomica comparativi del processo laterale Root Iniziazione Un&#39;applicazione della Lris è il confronto e correlazione dei profili di espressione genica durante la formazione delle radici laterali in specie diverse. Trascrittomica comparata approcci creare la possibilità di individuare i geni ortologhi coinvolti nel processo di sviluppo delle radici laterali in dive...

Watch this Scientific Journal Video about Lateral Root Inducible System in Arabidopsis and Maize at JoVE.com

Sistema inducibile della radice laterale nell'Arabidopsis e nel mais | Biologia | Video

Lateral Root Inducible System in Arabidopsis and Maize

Laterale Root inducibile di sistema in<em&gt; Arabidopsis</em&gt; E mais

The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is ...

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Lateral Root Inducible System in Arabidopsis and Maize

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13.6K Views.  Ghent. The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

Video: Lateral Root Inducible System in Arabidopsis and Maize

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

Preparazione e manutenzione dei neuroni dei gangli dorsali in Culture Compartmented

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

Ricerca

Biologia

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself dispositivo per il recupero di campioni crioconservati cadere accidentalmente nelle cisterne criogeniche

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Using Flatbed Scanners to Collect High-resolution Time-lapsed Images of the Arabidopsis Root Gravitropic Response

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories can face is the development and attainment of these methods. Observation of phenotypes in a process of interest is a typical objective of research labs studying gene function and this is often achieved through image capture. A particular process that is amenable to observation using imaging approaches is the corrective growth of a seedling root that has been displaced from alignment with the gravity vector. Imaging platforms used to measure the root gravitropic response can be expensive, relatively low in throughput, and/or labor intensive. These issues have been addressed by developing a high-throughput image capture method using inexpensive, yet high-resolution, flatbed scanners. Using this method, images can be captured every few minutes at 4,800 dpi. The current setup enables collection of 216 individual responses per day. The image data collected is of ample quality for image analysis applications.

This protocol describes a process for rapid collection of images of Arabidopsis seedlings responding to a gravity stimulus using commercially-available flatbed scanners. The method allows for inexpensive, high-volume capture of high-resolution images amenable for downstream analysis algorithms.

Utilizzo di Scanner Flatbed per la raccolta ad alta risoluzione e ritardi Immagini della Arabidopsis Root gravitropica risposta

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

Un test per Lateral Rigenerazione Line in Zebrafish adulti

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Misurazione spaziale e temporale Ca<sup&gt; 2 +</sup&gt; Segnali in Arabidopsis Piante

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Risoluzione Quantificazione alta del cristallino cellulosa accumulo in<em&gt; Arabidopsis</em&gt; Roots a Monitor tessuto-specifica delle cellule parete Modifiche

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

Micron-scala Phenotyping tecniche di mais fasci vascolari basato su x-Ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. Upon wounding of a leaf, an increase in cytosolic calcium ion concentration (Ca2+ signal) occurs at the wound site. This signal is rapidly transmitted to undamaged leaves, where defense responses are activated. Our recent research revealed that glutamate leaking from the wounded cells of the leaf into the apoplast around them serves as a wound signal. This glutamate activates glutamate receptor-like Ca2+ permeable channels, which then leads to long-distance Ca2+ signal propagation throughout the plant. The spatial and temporal characteristics of these events can be captured with real-time imaging of living plants expressing genetically encoded fluorescent biosensors. Here we introduce a plant-wide, real-time imaging method to monitor the dynamics of both the Ca2+ signals and changes in apoplastic glutamate that occur in response to wounding. This approach uses a wide-field fluorescence microscope and transgenic Arabidopsis plants expressing Green Fluorescent Protein (GFP)-based Ca2+ and glutamate biosensors. In addition, we present methodology to easily elicit wound-induced, glutamate-triggered rapid and long-distance Ca2+ signal propagation. This protocol can also be applied to studies on other plant stresses to help investigate how plant systemic signaling might be involved in their signaling and response networks.

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Extracellular glutamate-triggered systemic calcium signaling is critical for the induction of plant defense responses to mechanical wounding and herbivore attack in plants. This article describes a method to visualize the spatial and temporal dynamics of both these factors using Arabidopsis thaliana plants expressing calcium- and glutamate-sensitive fluorescent biosensors.

Imaging in tempo reale a ampio campo dei segnali di ferita locali e sistemici in Arabidopsis

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

Divergenza del microbiota radicale in diversi habitat in base a reti di correlazione ponderate

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...

A Simple Protocol for Mapping the Plant Root System Architecture Traits

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar tolerance to environmental challenges. An experimental protocol is presented for setting up the hydroponic system, plantlet growth, RSA spreading, and imaging. The approach used a magenta box-based hydroponic system containing polypropylene mesh supported by polycarbonate wedges. Experimental settings are exemplified by assessing the RSA of the plantlets under varying nutrient (phosphate [Pi]) supply. The system was established to examine the RSA of Arabidopsis, but it is readily adaptable to study other plants like Medicago sativa (Alfalfa). Arabidopsis thaliana (Col-0) plantlets are used in this investigation as an example to understand the plant RSA. Seeds are surface sterilized by treating ethanol and diluted commercial bleach, and kept at 4 &#176;C for stratification. The seeds are germinated and grown on a liquid half-MS medium on a polypropylene mesh supported by polycarbonate wedges. The plantlets are grown under standard growth conditions for the desired number days, gently picked out from the mesh, and submersed in water-containing agar plates. Each root system of the plantlets is spread gently on the water-filled plate with the help of a round art brush. These Petri plates are photographed or scanned at high resolution to document the RSA traits. The root traits, such as primary root, lateral roots, and branching zone, are measured using the freely available ImageJ software. This study provides techniques for measuring plant root characteristics in controlled environmental settings. We discuss how to (1) grow the plantlets, and collect and spread root samples, (2) obtain pictures of spread RSA samples, (3) capture the images, and (4) use image analysis software to quantify root attributes. The advantage of the present method is the versatile, easy, and efficient measurement of the RSA traits.

We use simple laboratory tools to examine the root system architecture (RSA) of Arabidopsis and Medicago. The plantlets are grown hydroponically over mesh and spread using an art brush to reveal the RSA. Images are taken using scanning or a high-resolution camera, then analyzed with ImageJ to map traits.

Un semplice protocollo per mappare i tratti dell'architettura del sistema di root delle piante

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar ...