This work was supported by the Natural Sciences and Engineering Council of Canada (NSERC), the Canada Research Chair (CRC) program, and the Canadian Foundation for Innovation (CFI).

<html> 1. coltura delle piante e preparazione <ol><li> Crescere piante di orzo idroponica per 7 giorni in una camera di crescita a clima controllato (per i dettagli, vedi 10). NOTA: E &#39;importante considerare l&#39;esame di piante in una varietà di fasi di sviluppo, come esigenze nutrizionali cambiano con l&#39;età. </li><li> Un giorno prima della sperimentazione, raggruppare diverse piantine insieme per fare una singola replica (3 piante per pacco per DI, 6 piante per pacco per CATE). Piantine Bundle avvolgendo un 2-cm pezzo di tubo Tygon attorno alla porzione basale dei germogli, e assicurare il tubo con del nastro adesivo per creare un &quot;collare&quot;. NOTA: Il numero di piante per pacco può variare in base alle condizioni sperimentali 10,13,14. Bundle viene fatto per migliorare le statistiche e precisione di misura, in particolare quando la massa radicale e / o attività specifica sono bassi. </li></ol> 2 Preparazione di Sperimentali Soluzioni / Materiali contenuti &quot;&gt; NOTA: La seguente è in genere eseguita 1 giorno prima sperimentazione. <ol><li> Per la DI, raccogliere le seguenti: Pre-etichettatura, etichettatura e soluzioni di desorbimento (per i dettagli, vedi 11), tubi di centrifugazione (per spin-essiccazione di campioni vegetali), e vial di campioni (per materiale vegetale e l&#39;attività specifica [S o; vedi sotto]). Aerare e mescolare tutte le soluzioni. </li><li> Per CATE, raccogliere le seguenti: soluzioni ben mescolato, etichettatura aerata e di eluizione (per i dettagli, vedi 10), imbuti, tubi di efflusso centrifugazione (per spin-essiccazione di campioni vegetali), e fiale di campioni (per eluati, campioni vegetali, e determinazione di S o fattore di diluizione e [D f; vedere sotto]). </li></ol> 3 Preparare radiotracciante ATTENZIONE: Le seguenti operazioni di sicurezza dovrebbero essere prese prima di lavorare con la radioattività. <ol><li> Assicurarsi che i requisiti del radioactiva licenza materiali sono compresi e seguiti. Indossare un equipaggiamento di sicurezza (ad esempio, occhiali, guanti, camice da laboratorio, piombo maglia / collare) e dosimetri (ad esempio, l&#39;anello TLD e distintivo). Impostazione di schermatura (cioè, plexiglas e mattoni di piombo) e svolgere un lavoro radioattivo dietro. Assicurarsi che un contatore Geiger-Müller è presente al fine di monitorare regolarmente per contaminazione. </li><li> Preparazione di 42 K + <ol><li> Porre un bicchiere pulito e asciutto sulla bilancia. Azzerare la bilancia. </li><li> Rimuovere fiala di tracciante (20 mCi di 42 K 2 CO 3, in forma di polvere) dalla confezione e versare tracciante nel becher. Prendere nota della massa. </li><li> Pipettare 19.93 ml di dH 2 O, seguita da 0,07 ml di H 2 SO 4, nel bicchiere. Questo guiderà la seguente reazione chimica: 42 K 2 CO 3 (S) + H 2 SO 4 (l) + H 2 O (l) → 42 </sup&gt; K 2 SO 4 (L) + CO 2 (v) + 2H 2 O (l) </li><li> Calcolare la concentrazione della soluzione madre radioattivi, data la massa e il peso molecolare di K 2 CO 3, e il volume (20 ml). NOTA: Se si lavora con 13 NH 3/13 NH 4 + Il tracciante è prodotto in un ciclotrone mediante il bombardamento protone dell&#39;atomo di ossigeno di acqua (tipicamente conseguente 100-200 mCi di attività, per dettagli di produzione, vedere 12). Poiché la quantità di 14 NH 3/14 NH 4 + è estremamente bassa in queste soluzioni, la concentrazione N della soluzione madre è trascurabile. </li></ol></li></ol> 4 Afflusso diretta (DI) di misura <ol><li> Per 42 K +, pipettare quantità di soluzione madre radioattivo necessario per raggiungere la concentrazione finale desiderata di K + nella soluzione di etichettatura. <ol><li> Per 13 NH 3/13 NH 4 +, pipetta una piccola quantità (&lt;0,5 ml) nella soluzione di etichettatura. Lasciare che la soluzione di etichettatura a mescolare bene (con aerazione). </li></ol></li><li> Pipettare un sotto-campione di 1 ml di soluzione di etichettatura in una provetta e ripetere tre volte (4 campioni in totale). <ol><li> Misurare la radioattività in flaconcini (in &quot;conteggi per minuto&quot;, CPM), utilizzando un contatore gamma. Verificare che il contatore è programmato in modo tale che le letture CPM sono corretti per il decadimento isotopico (questo è particolarmente importante per tali traccianti di breve durata). </li><li> Calcola S o (espressa come cpm mol -1), facendo la media dei conteggi dei quattro campioni (CPM ml -1) e dividendo per la concentrazione di substrato in soluzione (mmol ml -1). </li></ol></li><li> Immergere le radici in pre-etichettatura soluzione (non radioattivo) per 5 minuti, a pre-equilibrare le piante in condizioni di prova (vediad esempio, 10,13,14 per variazioni nel tempo di pre-label). </li><li> Immergere radici nella soluzione di etichettatura (radioattiva) per 5 min. NOTA: i tempi di etichettatura possono variare in base esperimento 3,4,7-10. </li><li> Trasferire le radici di soluzione desorbimento per 5 secondi per rimuovere la maggior parte della radioattività-aderente di superficie. Trasferire le radici in un secondo bicchiere di soluzione di desorbimento per 5 minuti a ulteriori chiare radici del tracciante extracellulare. </li><li> Sezionare e germogli separati, germogli basali e radici. </li><li> Mettere radici in provette da centrifuga e campioni di spin per 30 secondi in un a bassa velocità, clinico-grade centrifuga (~ 5.000 xg) per rimuovere la superficie e l&#39;acqua interstiziale. </li><li> Pesare radici (peso fresco, FW). </li><li> Contare la radioattività in campioni di piante (sparare, sparare basale, e la radice; vedere il punto 4.2.1). </li><li> Calcolare il flusso. Calcolare afflusso nella pianta utilizzando la formula Φ = Q * / S o WT L dove Φ è il flusso(G mol -1 h -1), Q * è la quantità di tracciante accumulata nei tessuti (cpm, di solito in radice, sparare e sparare basale, combinato), S o è l&#39;attività specifica della soluzione di etichettatura (cpm micromol - 1), w è il peso fresco radice (g), e L t è il tempo di etichettatura (hr). NOTA: il calcolo più sofisticato può essere fatto per tenere conto di simultanea tracciante efflusso dalle radici durante l&#39;etichettatura e desorbimento, in base ai parametri ottenuti da CATE (vedi sotto, per maggiori dettagli, vedi 4). </li></ol> 5. Compartimentale Analisi per Tracer Efflux (CATE) Misurazione <ol><li> Preparare la soluzione di etichettatura e misura S o (vedere i punti 4,1-4,2, sopra). </li><li> Misura fattore di diluizione (D f). NOTA: Spesso, la posizione del campione rispetto al rivelatore nel contatore gamma può influenzare la quantitàdi radiazione misurata. Vedi di discussione per i dettagli. <ol><li> Dopo aver misurato S o, aggiungere 19 ml di H 2 O per ogni campione (tale che il volume finale = volume eluito = 20 ml). Conteggio della radioattività in ogni campione da 20 ml (vedere il punto 4.2.1). </li><li> Calcolare D f dividendo il cpm medio dei campioni 1 ml dalla cpm media dei campioni di 20 ml. </li></ol></li><li> Immergere radici nella soluzione di etichettatura per 1 ora. </li><li> Rimuovere le piante da soluzione di etichettatura e di trasferimento piante per efflusso imbuto, assicurando tutto il materiale principale è dentro l&#39;imbuto. Delicatamente piante protetta a lato della efflusso imbuto applicando una piccola striscia di nastro adesivo sopra il collare di plastica. </li><li> Versare delicatamente il primo eluato nell&#39;imbuto. Avviare il timer (contando verso l&#39;alto). </li><li> Aprire il rubinetto e raccogliere l&#39;eluato nel flaconcino campione dopo 15 sec (nota: tempo di eluizione può variare, vedi sotto). Chiudere il rubinetto. Versare delicatamente il prossimo eluato nell&#39;imbuto. </li><li> Ripett passo 5.6 per il resto della serie eluizione, che segue, dal primo al eluato finale: 15 sec (quattro volte), 20 sec (tre volte), 30 sec (due volte), 40 sec (una volta), 50 sec (una volta), 1 min (25 volte), per un periodo di eluizione totale di 29,5 min NOTA: serie desorbimento può variare in base alle condizioni sperimentali 7-10,13,14. </li><li> Una volta che il protocollo eluizione è completo, piante raccolta (passi 4,6-4,8, sopra). </li><li> Contare la radioattività in eluati e campioni vegetali in gamma counter (moltiplicando la lettura per ciascun eluato da D f, vedi 5.2). </li><li> Rilascio della trama tracciante (cpm g (radice FW) -1 min -1) in funzione del tempo di eluizione. Per le condizioni di stato stazionario, eseguire regressioni lineari e calcoli di flussi, emivite di scambio, e le dimensioni della piscina (per i dettagli, vedere 6-9). </li></ol>

<ol>
	<li>Kronzucker, H. J., Coskun, D., Schulze, L. M., Wong, J. R., Britto, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+as+nutrient+and+toxicant.">Sodium as nutrient and toxicant.</a> Plant Soil. 369, 1-23 (2013).</li><li>Britto, D. T., Kronzucker, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NH4++toxicity+in+higher+plants:+a+critical+review.">NH4+ toxicity in higher plants: a critical review.</a> J. Plant Physiol. 159, 567-584 (2002).</li><li>Epstein, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+ion+absorption+by+roots.">Mechanism of ion absorption by roots.</a> Nature. 171, 83-84 (1953).</li><li>Britto, D. T., Kronzucker, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+unidirectional+influx+be+measured+in+higher+plants?+A+mathematical+approach+using+parameters+from+efflux+analysis.">Can unidirectional influx be measured in higher plants? A mathematical approach using parameters from efflux analysis.</a> New Phytol. 150, 37-47 (2001).</li><li>Britto, D. T., Kronzucker, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+of+potassium+transport+in+plants.">Cellular mechanisms of potassium transport in plants.</a> Physiol. Plant. 133, 637-650 (2008).</li><li>Walker, N. A., Pitman, M. G., L&#252;ttge, U., >Pitman, M. .. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+fluxes+across+membranes.">Measurement of fluxes across membranes.</a> Encyclopedia of plant physiology. 2 Part A, (1976).</li><li>Kronzucker, H. J., Siddiqi, M. Y., Glass, A. D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+13NH4++efflux+in+spruce+roots+-+A+test+case+for+phase+identification+in+compartmental+analysis.">Analysis of 13NH4+ efflux in spruce roots - A test case for phase identification in compartmental analysis.</a> Plant Physiol. 109, 481-490 (1995).</li><li>Siddiqi, M. Y., Glass, A. D. M., Ruth, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+the+uptake+of+nitrate+in+barley.+3.+Compartmentation+of+NO3-.">Studies of the uptake of nitrate in barley. 3. Compartmentation of NO3-.</a> J. Exp. Bot. 42, 1455-1463 (1991).</li><li>Lee, R. B., Clarkson, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrogen-13+studies+of+nitrate+fluxes+in+barley+roots.+1.+Compartmental+analysis+from+measurements+of+13N+efflux.">Nitrogen-13 studies of nitrate fluxes in barley roots. 1. Compartmental analysis from measurements of 13N efflux.</a> J. Exp. Bot. 37, 1753-1767 (1986).</li><li>Coskun, D., Britto, D. T., Kronzucker, H. J. <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+mechanism+of+potassium+release+from+barley+roots:+an+in+planta+42K++analysis.">Regulation and mechanism of potassium release from barley roots: an in planta 42K+ analysis.</a> New Phytol. 188, 1028-1038 (2010).</li><li>Britto, D. T., Kronzucker, H. J., Maathuis, F. .. J. .. M. .. ,. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluxes+measurements+of+cations+using+radioactive+tracers.">Fluxes measurements of cations using radioactive tracers.</a> Plant Mineral Nutrients: Methods and Protocols, Methods in Molecular Biology. Volume 953, 161-170 (2013).</li><li>Meeks, J. C., Knowles, R. ,., Blackburn, T. .. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=13N+techniques.">13N techniques.</a> Nitrogen isotope techniques. , 273-303 (1993).</li><li>Coskun, D., Britto, D. T., Li, M., Becker, A., Kronzucker, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+ammonia+gas+transport+accounts+for+futile+transmembrane+cycling+under+NH3/NH4++toxicity+in+plant+roots.">Rapid ammonia gas transport accounts for futile transmembrane cycling under NH3/NH4+ toxicity in plant roots.</a> Plant Physiol. 163, 1859-1867 (2013).</li><li>Coskun, D., Britto, D. T., Li, M., Oh, S., Kronzucker, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capacity+and+plasticity+of+potassium+channels+and+high-affinity+transporters+in+roots+of+barley+and+Arabidopsis.">Capacity and plasticity of potassium channels and high-affinity transporters in roots of barley and Arabidopsis.</a> Plant Physiol. 162, 496-511 (2013).</li><li>Johansson, I., 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=External+K++modulates+the+activity+of+the+Arabidopsis+potassium+channel+SKOR+via+an+unusual+mechanism.">External K+ modulates the activity of the Arabidopsis potassium channel SKOR via an unusual mechanism.</a> Plant J. 46, 269-281 (2006).</li><li>Nocito, F. F., Sacchi, G. A., Cocucci, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+depolarization+induces+K++efflux+from+subapical+maize+root+segments.">Membrane depolarization induces K+ efflux from subapical maize root segments.</a> New Phytol. 154, 45-51 (2002).</li><li>Wang, M. Y., Glass, A. D. M., Shaff, J. E., Kochian, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ammonium+uptake+by+rice+roots.+3.+Electrophysiology.">Ammonium uptake by rice roots. 3. Electrophysiology.</a> Plant Physiol. 104, 899-906 (1994).</li><li>Walker, D. J., Leigh, R. A., Miller, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+homeostasis+in+vacuolate+plant+cells.">Potassium homeostasis in vacuolate plant cells.</a> Proc. Natl. Acad. Sci. U.S.A. 93, 10510-10514 (1996).</li><li>Holm, L. 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=NH3+and+NH4++permeability+in+aquaporin-expressing+Xenopus+oocytes.">NH3 and NH4+ permeability in aquaporin-expressing Xenopus oocytes.</a> Pflugers Archiv. Eur. J. Physiol. 450, 415-428 (2005).</li><li>Britto, D. T., Kronzucker, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-stimulation+of+13NH4++efflux+provides+evidence+for+the+cytosolic+origin+of+tracer+in+the+compartmental+analysis+of+barley+roots.">Trans-stimulation of 13NH4+ efflux provides evidence for the cytosolic origin of tracer in the compartmental analysis of barley roots.</a> Funct. Plant Biol. 30, 1233-1238 (2003).</li><li>Malagoli, P., Britto, D. T., Schulze, L. M., Kronzucker, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Futile+Na++cycling+at+the+root+plasma+membrane+in+rice+(Oryza+sativa+L.):+kinetics,+energetics,+and+relationship+to+salinity+tolerance.">Futile Na+ cycling at the root plasma membrane in rice (Oryza sativa L.): kinetics, energetics, and relationship to salinity tolerance.</a> J. Exp. Bot. 59, 4109-4117 (2008).</li><li>Kronzucker, H. J., Britto, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+transport+in+plants:+a+critical+review.">Sodium transport in plants: a critical review.</a> New Phytol. 189, 54-81 (2011).</li></ol>

The authors have nothing to disclose.

Come dimostrato negli esempi precedenti, il metodo radiofarmaco è un potente mezzo di misurare flussi unidirezionali di nutrienti e sostanze tossiche in planta. Figura 1 mostra che NH 3 afflusso può raggiungere superiore a 225 micromol g -1 h -1, che è forse la più alto bona fide flusso transmembrana mai riportato in un sistema impiantistico 13, ma la grandezza di questo flusso non sarebbe visibile se sono stati misurati solo i flussi net...

L&#39;assorbimento e la distribuzione di nutrienti e sostanze tossiche influenzano fortemente la crescita delle piante. Di conseguenza, l&#39;indagine dei processi di trasporto sottostanti costituisce un&#39;importante area di ricerca in biologia vegetale e scienze agrarie 1,2, soprattutto nei contesti di ottimizzazione nutrizionale e stress ambientali (ad esempio, stress salino, la tossicità di ammonio). Primo fra i metodi per la misurazione dei flussi nelle piante è l&#39;uso di traccianti radioisotopici, che è stato sviluppato in modo significativo nel 1950 (si veda ad esempio, 3) e continua ad essere ampiamente usato oggi. Altri metodi, come la misurazione di esaurimento degli elementi nutritivi dal mezzo di root e / o l&#39;accumulo nei tessuti, l&#39;uso di microelettrodi vibranti ione-selettivi, come MIFE (microelettrodo ion stima di flusso) e SIET (scansione tecnica elettrodo ione-selettivo), e l&#39;uso di coloranti fluorescenti ione-selettivo, sono anche ampiamente applicati, ma sono limitati nella loro capacità di rilevare l&#39;influenza nettassi (cioè, la differenza tra afflusso e l&#39;efflusso). L&#39;uso di radioisotopi, dall&#39;altro, consente al ricercatore la capacità unica di isolare e quantificare flussi unidirezionali, che può essere utilizzato per risolvere parametri cinetici (ad esempio, K M e V max), e fornire una conoscenza della capacità, energetica, meccanismi e regolazione, dei sistemi di trasporto. Misure di flusso unidirezionali realizzati con radiotraccianti sono particolarmente utili in condizioni in cui il flusso nella direzione opposta è alto, e il fatturato delle piscine intracellulari è rapida 4. Inoltre, i metodi radiotracciante consentono misurazioni condotte sotto concentrazioni piuttosto elevate di substrato, a differenza di molte altre tecniche (vedi &#39;Discussione&#39;, qui di seguito), perché l&#39;isotopo tracciato si osserva su uno sfondo di un altro isotopo dello stesso elemento. Qui, forniamo la procedura dettagliata per la misurazione di radioisotopi unidirezionale e net flussi di nutrienti minerali e di sostanze tossiche nelle piante intatte. L&#39;accento verrà effettuato sulla misurazione del flusso di potassio (K +), un impianto di macronutrienti 5, e ammoniaca / ammonio (NH 3 / NH 4 +), un&#39;altra macronutrienti che è, tuttavia, tossici se presenti a concentrazioni elevate (ad esempio, 1- 10 mM) 2. Useremo i radioisotopi 42 K + (t mezzo = 12.36 ore) e 13 NH 3/13 NH 4 + (t 1/2 = 9,98 min), rispettivamente in piantine intatte del orzo sistema modello (Hordeum vulgare L .), nella descrizione dei due protocolli principali: afflusso diretta (DI) e l&#39;analisi compartimentale da tracciante efflusso (CATE). Dobbiamo notare fin dall&#39;inizio che questo articolo descrive semplicemente i passaggi necessari per eseguire ogni protocollo da. Se del caso, brevi spiegazioni dei calcoli e la teoria sono forniti, ma dettagliate esposizioni di ogni tecnica&#39;S sfondo e la teoria si possono trovare in diversi articoli importanti sull&#39;argomento 4,6-9. È importante sottolineare che questi protocolli sono ampiamente trasferibili ad flux analisi di altri nutrienti / sostanze tossiche (ad esempio, 24 Na +, 22 Na +, Rb + 86, 13 NO 3 -) e di altre specie di piante, anche se con qualche distinguo (vedi sotto) . Sottolineiamo inoltre l&#39;importanza che tutti i ricercatori che lavorano con materiali radioattivi devono lavorare sotto una licenza organizzato tramite ionizzanti regolatore di sicurezza di radiazione della loro istituzione.

<table border="0" cellpadding="0" cellspacing="0" width="605">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:103px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gamma counter</td>
			<td style="width:103px;">Perkin Elmer</td>
			<td style="width:119px;"></td>
			<td>Model: Wallac 1480 Wizard 3&#34;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Geiger-M&#252;ller counter</td>
			<td style="width:103px;">Ludlum Measurements Inc.</td>
			<td style="width:119px;"></td>
			<td>Model 3 survey meter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">400-mL glass beakers</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">89000-206</td>
			<td>For pre-absorption, absorption, and desorption solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass funnel</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">89000-466</td>
			<td>For efflux funnel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Large tubing</td>
			<td style="width:103px;">VWR</td>
			<td align="right" style="width:119px;">529297</td>
			<td>For efflux funnel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Medium tubing</td>
			<td style="width:103px;">VWR</td>
			<td align="right" style="width:119px;">684783</td>
			<td>For bundling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Small tubing</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">63013-541</td>
			<td>For aeration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Aeration manifold</td>
			<td style="width:103px;">Penn Plax Air Tech</td>
			<td style="width:119px;">vat 5.5</td>
			<td>To control/distribute pressurized air into solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass scintillation vials</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">66022-128</td>
			<td>For gamma counting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass centrifuge tubes</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">47729-576</td>
			<td>For spin-drying root samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Kimwipes</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">470173-504</td>
			<td>For spin-drying root samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissecting scissors</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">470001-828</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forceps</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">470005-496</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Low-speed clinical centrifuge</td>
			<td style="width:103px;">International Equipment Co.</td>
			<td style="width:119px;">76466M-4</td>
			<td>For spin-drying root samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1-mL pipette</td>
			<td style="width:103px;">Gilson</td>
			<td style="width:119px;">F144493</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10-mL pipette</td>
			<td style="width:103px;">Gilson</td>
			<td style="width:119px;">F144494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1-mL pipette tips</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">89079-470</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10-mL pipette tips</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">89087-532</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Analytical balance</td>
			<td style="width:103px;">Mettler toledo</td>
			<td style="width:119px;">PB403-S/FACT</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring fluxes of mineral nutrients and toxicants in plants with radioactive tracers

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. Radioisotope tracing is a major technique used to measure such fluxes, both within plants, and between plants and their environments. Flux data obtained with radiotracer protocols can help elucidate the capacity, mechanism, regulation, and energetics of transport systems for specific mineral nutrients or toxicants, and can provide insight into compartmentation and turnover rates of subcellular mineral and metabolite pools. Here, we describe two major radioisotope protocols used in plant biology: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We focus on flux measurement of potassium (K+) as a nutrient, and ammonia/ammonium (NH3/NH4+) as a toxicant, in intact seedlings of the model species barley (Hordeum vulgare L.). These protocols can be readily adapted to other experimental systems (e.g., different species, excised plant material, and other nutrients/toxicants). Advantages and limitations of these protocols are discussed.

La figura 1 mostra isoterme trovati con la tecnica DI (con 13 N), per l&#39;afflusso di NH 3 in radici di piante di orzo coltivate intatte ad alta (10 mm) NH 4 +, e sia bassa (0,02 mm) o alto (5 mM ) K +. Le isoterme mostrano cinetica di Michaelis-Menten quando NH 3 flussi sono riportati in grafico in funzione della concentrazione esterna NH 3 ([NH 3] ext; rettificato dalla variazione del pH della soluzione 13)....

Watch this Scientific Journal Video about Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers at JoVE.com

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

Flussi di minerali nutrienti e sostanze tossiche in misura Piante con traccianti radioattivi

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

measuring-fluxes-mineral-nutrients-toxicants-plants-with-radioactive

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11.2K Views.  University of Toronto. In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

Video: Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as&#160;fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

Espressione ricombinante Cellulase Cel5A da<em&gt; Trichoderma reesei</em&gt; In piante di tabacco

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture ...

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Measuring Rates of Herbicide Metabolism in Dicot Weeds with an Excised Leaf Assay

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we developed an excised leaf assay combined with a vegetative cloning strategy to normalize herbicide uptake and remove translocation as contributing factors in herbicide-resistant (R) and &#8211;sensitive (S) waterhemp populations. Biokinetic analyses of organic pesticides in plants typically include the determination of uptake, translocation (delivery to the target site), metabolic fate, and interactions with the target site. Herbicide metabolism is an important parameter to measure in herbicide-resistant weeds and herbicide-tolerant crops, and is typically accomplished with whole-plant tests using radiolabeled herbicides. However, one difficulty with interpreting biokinetic parameters derived from whole-plant methods is that translocation is often affected by rates of herbicide metabolism, since polar metabolites are usually not mobile within the plant following herbicide detoxification reactions. Advantages of the protocol described in this manuscript include reproducible, accurate, and rapid determination of herbicide degradation rates in R and S populations, a substantial decrease in the amount of radiolabeled herbicide consumed, a large reduction in radiolabeled plant materials requiring further handling and disposal, and the ability to perform radiolabeled herbicide experiments in the lab or growth chamber instead of a greenhouse. As herbicide resistance continues to develop and spread in dicot weed populations worldwide, the excised leaf assay method developed and described herein will provide an invaluable technique for investigating non-target site-based resistance due to enhanced rates of herbicide metabolism and detoxification.

This manuscript describes how herbicide metabolism rates can be effectively quantified with excised leaves from a dicot weed, thereby reducing variability and removing any possible confounding effects of herbicide uptake or translocation typically observed in whole-plant assays.

Misurare Tassi di erbicida Metabolismo in Dicot Erbacce con un saggio asportati Foglia

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we ...

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in nutrient and gas cycles. After cores with intact sediment-water interfaces are collected, they are submerged in incubation tanks and kept under aerobic conditions at in situ temperatures. To initiate a time course of overlying water chemistry, cores are sealed without bubbles using a top cap with a suspended stirrer. Time courses of 4-7 sample points are used to determine the rate of sediment water exchange. Artificial illumination simulates day-time conditions for shallow photosynthetic sediments, and in conjunction with dark incubations can provide net exchanges on a daily basis. The net measurement of N2 is made possible by sampling a time course of dissolved gas concentrations, with high precision mass spectrometric analysis of N2:Ar ratios providing a means to measure N2 concentrations. We have successfully applied this approach to lakes, reservoirs, estuaries, wetlands and storm water ponds, and with care, this approach provides valuable information on biogeochemical balances in aquatic ecosystems.

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

Here, we present small core incubations for the measurement of sediment-water gas and solute exchange. These will provide reliable measurements of sediment-water exchange that assess the role of sediment in influencing biological and biogeochemical processes in aquatic ecosystems.

La Borsa bentonici di O<sub&gt; 2</sub&gt;, N<sub&gt; 2</sub&gt; e nutrienti disciolti Utilizzo piccolo nucleo Incubazioni

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in ...

Continuous Instream Monitoring of Nutrients and Sediment in Agricultural Watersheds

Pollutant concentrations and loads in watersheds vary considerably with time and space. Accurate and timely information on the magnitude of pollutants in water resources is a prerequisite for understanding the drivers of the pollutant loads and for making informed water resource management decisions. The commonly used &#34;grab sampling&#34; method provides the concentrations of pollutants at the time of sampling (i.e., a snapshot concentration) and may under- or overpredict the pollutant concentrations and loads. Continuous monitoring of nutrients and sediment has recently received more attention due to advances in computing, sensing technology, and storage devices. This protocol demonstrates the use of sensors, sondes, and instrumentation to continuously monitor in situ nitrate, ammonium, turbidity, pH, conductivity, temperature, and dissolved oxygen (DO) and to calculate the loads from two streams (ditches) in two agricultural watersheds. With the proper calibration, maintenance, and operation of sensors and sondes, good water quality data can be obtained by overcoming challenging conditions such as fouling and debris buildup. The method can also be used in watersheds of various sizes and characterized by agricultural, forested, and/or urban land.

With the advancement of technology and the rise in end-user expectations, the need and use of higher temporal resolution data for pollutant load estimation has increased. This protocol describes a method for continuous in situ water quality monitoring to obtain higher temporal resolution data for informed water resource management decisions.

Instream continuo monitoraggio delle sostanze nutrienti e sedimenti nei bacini agricoli

Pollutant concentrations and loads in watersheds vary considerably with time and space. Accurate and timely information on the magnitude of pollutants in ...

Assessment of Methane and Nitrous Oxide Fluxes from Paddy Field by Means of Static Closed Chambers Maintaining Plants Within Headspace

This protocol describes the measurement of greenhouse gas (GHG) emissions from paddy soils using the static closed chamber technique. This method is based on the diffusion theory. A known volume of air overlaying a defined soil area is enclosed within a parallelepiped cover (named &#34;chamber&#34;), for a defined period of time. During this enclosure period, gases (methane (CH4) and nitrous oxide (N2O)) move from soil pore air near their microbial source (i.e., methanogens, nitrifiers, denitrifiers) to the chamber headspace, following a natural concentration gradient. Fluxes are then estimated from chamber headspace concentration variations sampled at regular intervals throughout the enclosure and then analyzed with gas chromatography. Among the techniques available for GHG measurement, the static closed chamber method is suitable for plot experiments, as it does not require large homogenously treated soil areas. Furthermore, it is manageable with limited resources and can identify relationships among ecosystem properties, processes, and fluxes, especially when combined with GHG driving force measurements. Nevertheless, with respect to the micrometeorological method, it causes a minimal but still unavoidable soil disturbance, and allows a minor temporal resolution. Several phases are key to the method implementation: i) chamber design and deployment, ii) sample handling and analyses, and iii) flux estimation. Technique implementation success in paddy fields demands adjustments for field flooding during much of the cropping cycle, and for rice plant maintenance within the chamber headspace during measurements. Therefore, the additional elements to be considered with respect to the usual application of non-flooded agricultural soils consist of devices for: i) avoiding any unintended water disturbance that could overestimate fluxes, and ii) including rice plants within the chamber headspace to fully consider gases emitted through aerenchyma transportation.

The overall goal of this protocol is to measure greenhouse gas emissions from paddy fields using the static closed chamber technique. The measurement system needs specific adjustments due to the presence of both a permanent water layer in the field and of the plants within the chamber headspace.

Valutazione di metano e protossido di azoto flussi da risaia per mezzo di statica chiuso Chambers mantenendo piante all'interno dello spazio di testa

This protocol describes the measurement of greenhouse gas (GHG) emissions from paddy soils using the static closed chamber technique. This method is based ...

Protocol for Producing Three-Dimensional Infrared Video of Freezing in Plants

Freezing in plants can be monitored using infrared (IR) thermography, because when water freezes, it gives off heat. However, problems with color contrast make 2-dimensions (2D) infrared images somewhat difficult to interpret. Viewing an IR image or the video of plants freezing in 3 dimensions (3D) would allow a more accurate identification of sites for ice nucleation as well as the progression of freezing. In this paper, we demonstrate a relatively simple means to produce a 3D infrared video of a strawberry plant freezing. Strawberry is an economically important crop that is subjected to unexpected spring freeze events in many areas of the world. An accurate understanding of the freezing in strawberry will provide both breeders and growers with more economical ways to prevent any damage to plants during freezing conditions.
The technique involves a positioning of two IR cameras at slightly different angles to film the strawberry freezing. The two video streams will be precisely synchronized using a screen capture software that records both cameras simultaneously. The recordings will then be imported into the imaging software and processed using an anaglyph technique. Using red-blue glasses, the 3D video will make it easier to determine the precise site of ice nucleation on leaf surfaces.

Here, we present a protocol to image a strawberry plant freezing in 3 dimensions. Two infrared cameras positioned at slightly different angles are used to produce a red-blue anaglyph video to observe the freezing of the plant in 3 dimensions.

Protocollo per la produzione di Video tridimensionale a raggi infrarossi di congelamento in piante

Freezing in plants can be monitored using infrared (IR) thermography, because when water freezes, it gives off heat. However, problems with color contrast ...

An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive harvest or expensive equipment. We present a customizable and affordable rhizobox method that allows the non-destructive visualization of root growth over time and is particularly well-suited to studying root plasticity in response to localized resource patches. The method was validated by assessing maize genotypic variation in plasticity responses to patches containing 15N-labeled legume residue. Methods are described to obtain representative developmental measurements over time, measure root length density in resource-containing and control patches, calculate root growth rates, and determine 15N recovery by plant roots and shoots. Advantages, caveats, and potential future applications of the method are also discussed. Although care must be taken to ensure that experimental conditions do not bias root growth data, the rhizobox protocol presented here yields reliable results if carried out with sufficient attention to detail.

Visualizing and measuring root growth in situ is extremely challenging. We present a customizable rhizobox method to track root development and proliferation over time in response to nutrient enrichment. This method is used to analyze maize genotypic differences in root plasticity in response to an organic nitrogen source.

Un protocollo di Rhizobox ottimizzato per visualizzare la crescita delle radici e tempi di risposta alle sostanze nutrienti localizzate

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive ...

Measuring the Shape and Size of Activated Sludge Particles Immobilized in Agar with an Open Source Software Pipeline

Experimental bioreactors, such as those treating wastewater, contain particles whose size and shape are important parameters. For example, the size and shape of activated sludge flocs can indicate the conditions at the microscale, and also directly affect how well the sludge settles in a clarifier.
Particle size and shape are both misleadingly &#39;simple&#39; measurements. Many subtle issues, often unaddressed in informal protocols, can arise when sampling, imaging, and analyzing particles. Sampling methods may be biased or not provide enough statistical power. The samples themselves may be poorly preserved or undergo alteration during immobilization. Images may not be of sufficient quality; overlapping particles, depth of field, magnification level, and various noise can all produce poor results. Poorly specified analysis can introduce bias, such as that produced by manual image thresholding and segmentation.
Affordability and throughput are desirable alongside reproducibility. An affordable, high throughput method can enable more frequent particle measurement, producing many images containing thousands of particles. A method that uses inexpensive reagents, a common dissecting microscope, and freely-available open source analysis software allows repeatable, accessible, reproducible, and partially-automated experimental results. Further, the product of such a method can be well-formatted, well-defined, and easily understood by data analysis software, easing both within-lab analyses and data sharing between labs.
We present a protocol that details the steps needed to produce such a product, including: sampling, sample preparation and immobilization in agar, digital image acquisition, digital image analysis, and examples of experiment-specific figure generation from the analysis results. We have also included an open-source data analysis pipeline to support this protocol.

The size and shape of particles in activated sludge are important parameters that are measured using varying methods. Inaccuracies arise from non-representative sampling, suboptimal images, and subjective analysis parameters. To minimize these errors and ease measurement, we present a protocol specifying every step, including an open source software pipeline.

Misurare la forma e la dimensione delle particelle di fango attivo immobilizzato in Agar con una Pipeline di Software Open Source

Experimental bioreactors, such as those treating wastewater, contain particles whose size and shape are important parameters. For example, the size and ...

Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water distribution in plant tissues in combination with freeze fixation techniques using liquid nitrogen (LN2). For woody species, however, preparations for observing the xylem transverse-cut surface involve some difficulties due to the orientation of wood fibers. Additionally, higher tension in the water column in xylem conduits can occasionally cause artifactual changes in water distribution, especially during sample fixation and collection. In this study, we demonstrate an efficient procedure to observe the water distribution within the xylem of woody plants in situ&#160;by using a cryostat and cryo-SEM. At first, during sample collection, measuring the xylem water potential should determine whether high tension is present in the xylem conduits. When the xylem water potential is low (&#60; ca. &#8722;0.5 MPa), a tension relaxation procedure is needed to facilitate better preservation of the water status in xylem conduits during sample freeze fixation. Next, a watertight collar is attached around the tree stem and filled with LN2&#160;for freeze fixation of the water status of xylem. After harvesting, care should be taken to ensure that the sample is preserved frozen while completing the procedures of sample preparation for observation. A cryostat is employed to clearly expose the xylem transverse-cut surface. In cryo-SEM observations, time adjustment for freeze-etching is required to remove frost dust and accentuate the edge of the cell walls on the viewing surface. Our results demonstrate the applicability of cryo-SEM techniques for the observation of water distribution within xylem at cellular and subcellular levels. The combination of cryo-SEM with non-destructive in situ observation techniques will profoundly improve the exploration of woody plant water flow dynamics.

Observing the water distribution within the xylem provides significant information regarding water flow dynamics in woody plants. In this study, we demonstrate the practical approach to observe xylem water distribution in situ&#160;by using a cryostat and cryo-SEM, which eliminates artifactual changes in the water status during sample preparation.

Distribuzione dell'acqua Xylem nelle piante legnose con un microscopio elettronico a scansione crio-scansione

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water ...

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term effects are very important in judging their consequences in the environment and on human health. In demonstrating long-term influences, effects of chemicals over generations in recent studies provide new insight. Here, we describe protocols for studying effects of chemicals over multiple generations using free-living nematode Caenorhabditis elegans. Two aspects are presented: (1) trans-generational (TG) and (2) multi-generational effect studies, the latter of which is separated to multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. The TG effect study is robust with a simple purpose to determine whether chemical exposure to parents can result in any residual consequences on offspring. After the effects are measured on parents, sodium hypochlorite solutions are used to kill the parents and keep the offspring so as to facilitate effect measurement on the offspring. The TG effect study is used to determine whether the offspring are affected when their parent is exposed to the pollutants. The MGE and MGR effect study is systematical used to determine whether continuous generational exposure can result in adaptive responses in offspring over generations. Careful pick-up and transfer are used to distinguish generations to facilitate effect measurement on each generation. We also combined protocols to measure locomotion behavior, reproduction, lifespan, biochemical and gene expression changes. Some example experiments are also presented to illustrate the trans- and multi-generational effect studies.

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

Utilizzo di Caenorhabditis elegans per studiare gli effetti trans e multigenerazionali dei tossici

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...