This work was supported by Biotech Risk Assessment Grant Program competitive grand no. 2006-39454-17438 to A. Snow, K. Mercer, and H. Alexander from the United States Department of Agriculture, National Institute of Food and Agriculture. Experiments using this method were conducted at and supported by the University of Kansas Field Station, a research unit of the Kansas Biological Survey and the University of Kansas. The authors would like to thank P. Jourdan and E. Regnier for helpful reviews on earlier versions of this manuscript. Additionally, this work was aided by the contributions of the staff at the University of Kansas Field Station, Waterman Farm at the Ohio State University (OSU), the USDA Ornamental Plant Germplasm Center at OSU, and the Seed Biology Lab in the Department of Horticulture and Crop Science at OSU, especially E. Renze, S. Stieve, A. Evans, and E. Grassbaugh, for technical support.

1. Raccogliere seme da più specie o Croci controllata di una singola specie Nota: Questo esempio del seme utilizzato da 15 tipi incrociate all&#39;interno della annuus specie Helianthus (girasole) utilizzando selvaggio, ibrido, e tipi di colture come il genitore materno (che produce seme). <ol><li> Alla fine della stagione di crescita, raccogliere teste di semi maturi in sacchetti etichettati. Pulire seme dal loglio e luogo di semi in buste etichettati con dei genitori a croce in formato standard (ad esempio, materno x paterna). </li><li> seme di massa insieme in grandi buste utilizzando la stessa quantità di seme da un numero uguale di identici tipi trasversali dei genitori. Ad esempio, 100 semi da 10 tipi incrociate con gli stessi genitori materni e paterni. Etichettare le buste con l&#39;identificazione (tipo incrociato) le informazioni. Queste buste grandi sono maestri di sementi da utilizzare nella compilazione strisce sepoltura nella sezione 2. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody&gt; <tr><td></td><td colspan="3"> Parent materna </td></tr><tr><td> Parent Paternal </td><td> Wild: wXw </td><td> F 1: WXC </td><td> Crop: CxC </td></tr><tr><td> Wild: wXw </td><td> 0% wXw </td><td> 25% F 1 xW † </td><td> 50% CXW † </td></tr><tr><td> BC: WxF1 o F1xW </td><td> 12,5% WxBC </td><td> 37,5% F 1 XBC </td><td> 62,5% CxBC </td></tr><tr><td> F 1: WXC </td><td> 25% wxf 1 † </td><td> 50% F 1 xF 1 </td><td> 75% CxF 1 † </td></tr><tr><td> F 2: F 1 xF 1 </td><td> 25% wxf 2 </td><td> 50% F 1 xF 2 </td><td> 75% CxF 2 </td></tr><tr><td> Crop: CxC </td><td> 50% WXC † </td><td> 75% F 1 xC † </td><td> 100% CxC </td></tr></tbody></table> Tabella 1. parentale croce tIPI prodotte dalla mano impollinazione. girasole tipi incrociati ibride colture-wild sono state prodotte con la mano l&#39;impollinazione per l&#39;uso in esperimento di semi di sepoltura. Per tutti i tipi di fondo, il genitore materno elencato per primo e il genitore paterna elencato secondo. tipi Croce contrassegnati con † fanno parte di reciproci coppie di tipo trasversale con gli stessi alleli% del raccolto, ma diversi genitori materni. La tabella è stata precedentemente pubblicata in: Pace, BA et al. (2015) 15. 2. Creare personalizzate strisce Burial Seed NOTA: In questo esempio, abbiamo avuto tre trattamenti data di uscita e 15 replicati, quindi abbiamo richiesto 45 strisce totale. Questo esempio utilizza 15 scomparti per striscia così, abbiamo richiesto abbastanza tessuto di poliestere a maglia fine o zanzariere per ospitare 15, 7 x 10 cm scomparti. Vedere la Figura 1. <ol><li> Inizia dal taglio di una striscia di 20 x 105 centimetri di tessuto per ogni striscia di semi. </li><li> Misurare e segnare scomparti, quindi piegare il tessuto a metà. Utilizzando un higpistola per colla h-temperatura, guarnizione uno corto (10 cm) fine. Una volta che la colla si raffredda leggermente, ma non completamente, premere i lati del tessuto insieme per creare una tenuta. ATTENZIONE: ad alta temperatura colla e colla punte pistola può provocare ustioni se il contatto con la pelle è prolungata. </li><li> linee di colla dal bordo ripiegato della breve estremità aperta ad ogni segno di comparto. Premere le due parti di tessuto insieme lungo le linee di colla per garantire guarnizioni senza fori tra i compartimenti. I semi che migrano tra i compartimenti contribuiscono a errore sperimentale. NOTA: Ora che sono stati creati i comparti, il bordo spiegato dovrebbe rimanere aperto e pronto a ricevere semi e le loro informazioni di identificazione (nome croce tipo). </li><li> Casualmente assegnare i tipi di croce seme di scomparti per ogni striscia. <ol><li> Riempire compartimenti seme con un unico numero di semi (20 per ciascun comparto sono utilizzati in questo esempio). Per le etichette, usare marcatori permanenti industriali sul cut-to-fit freccette da giardino in plastica in modo che siano in grado diresistere sepoltura. NOTA: i metodi di etichettatura alternative possono essere utilizzati. </li><li> Dopo ogni scomparto è riempita con semi e la sua etichetta, chiudere il vano immediatamente con la pistola colla ad alta temperatura. Proseguire fino a quando l&#39;intera striscia di seme è completamente sigillato. Etichettare striscia complessiva nel primo compartimento con replicare, blocco e / o informazioni sul trattamento. NOTA: Per il nostro studio, dato che abbiamo avuto traslochi temporali, blocchi, e si replicano le strisce all&#39;interno di blocchi, abbiamo incluso tutte queste informazioni. </li><li> Verificare la presenza di spazi tra compartimenti e colla posto come necessario prima di passare al successivo strip. </li></ol></li></ol><img alt="Figura 1" src="/files/ftp_upload/54663/54663fig1.jpg" /> Figura 1. sepoltura striscia seme schematica. Esempio di striscia sepoltura mostrando identificazione del tipo di croce per i singoli comparti. genitore materno è nominata per prima con il genitore paterna elencati secondo. I colori indicano diversa discendenza materna,con il giallo per il selvaggio, blu per F 1 ibrido, e rosso per il raccolto. <a href="http://ecsource.jove.com/files/ftp_upload/54663/54663fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> 3. Bury strisce seme nel campo <ol><li> Scavare trame alla profondità specificata per le strisce di casa. Assegnare strisce a singoli blocchi in cui strisce sono disposte in modo casuale per consentire la data di uscita da utilizzare come trama principale e l&#39;identità vano (in questo esempio, di tipo incrociato) come sottotrama in analisi. </li><li> Coprire le strisce con la terra. Tagliare il metallo panno hardware di maglie, da 10 cm più grande di trame e posto oltre le strisce sepolti. Utilizzare U-pin per proteggere i bordi e gli spigoli della copertura del panno con la terra. NOTA: Questo rivestimento è di escludere che scavano i predatori di semi. Come ulteriore opzione, Mercer et al. 13 costruito e scatole di stoffa hardware che circondavano le strisce di sementi a causa della forte pressione Gopher nel loro campo loca sepoltizione.) </li><li> Mettere bandiere di geometra colorato per distinguere posizione strip e rimozione date per completare record chiaro scritte. </li></ol> 4. Scavare strisce e valutare Seed <ol><li> Scavare le strisce di semi assegnati a una certa data rimozione. Mantenere le strisce in un ambiente umido fresco fino a raggiungere il laboratorio in cui la valutazione del seme avrà luogo. Questo può essere realizzato avvolgendo strisce in giornali bagnati e metterli in un dispositivo di raffreddamento per il trasporto o il trasporto di notte. </li><li> Risciacquare fango e terreno da strisce in acqua fino a quando le strisce sono abbastanza pulite che i detriti esterni saranno non oscuri i semi. </li><li> Usare le forbici per tagliare aperto uno scomparto alla volta. Conte semi germinati e rimuoverli. Conte semi chiaramente morti (ad esempio, Marcio, o semi putrefatti). Mettere i semi non germinati in etichetta, carta assorbente umida Petri dimensioni per distinguere ulteriormente i semi non germinati, dormienti, e morti. <ol><li> Posizionare piastre di Petri con non germinato schiacciatosemi in un set di camera di crescita per le condizioni di germinazione standard: 25 ° C / 10 ° C 12 ore giorno / notte. Consentire semi una settimana di germinare. Qualsiasi che, contare questi come valida, ma non germinati dal momento che non germinano in condizioni di campo. </li></ol></li><li> Dopo il trattamento camera di crescita, prova rimanendo semi non germinati per la vitalità usando Tetrazolium cloruro (TZ) 13. Per fare questo, costituiscono una soluzione per cento di TZ adatti alla specie di studio utilizzando il AOSA Tetrazolium Testing Handbook 14. Per questo esempio, si usa una soluzione 1%. <ol><li> Tagliare i semi non germinati a metà e porre una metà erano l&#39;embrione e endosperma sono visibili in una capsula di Petri contenente soluzione abbastanza TZ per coprire il seme (semi possono galleggiare, questo è bene). </li><li> Posizionare i piatti TZ Petri in un incubatore a 30 ° C e attendere 3 ore. Nota: Le temperature più elevate si ottengono una reazione più veloce, se desiderato, ma overstaining è un rischio. </li></ol></li> &lt;li&gt; Rimuovere piastre di Petri dal termostato e valutare. I semi che si sono macchiati rosso nei loro embrioni sono germinabili, mentre i semi non colorati sono morti. Consultare il manuale Tetrazolium Testing AOSA per maggiori dettagli su casi marginali distintivi e particolari specie. </li></ol>

<ol>
	<li>Walck, J. L., Baskin, J. M., Baskin, C. C., Hidayati, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+transient+and+persistent+seed+banks+in+species+with+pronounced+seasonal+dormancy+and+germination+patterns.">Defining transient and persistent seed banks in species with pronounced seasonal dormancy and germination patterns.</a> Seed Sci Res. 15 (3), 189-196 (2005).</li><li>Alexander, H. A., Schrag, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+soil+seed+banks+and+newly+dispersed+seeds+in+population+dynamics+of+the+annual+sunflower.+Helianthus+annuus.">Role of soil seed banks and newly dispersed seeds in population dynamics of the annual sunflower. Helianthus annuus.</a> J Ecol. 91, 987-998 (2003).</li><li>Burnside, O. C., Wilson, R. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+classification+system+for+seed+dormancy.">A classification system for seed dormancy.</a> Seed Sci Res. 14 (1), 1-16 (2004).</li><li>Donohue, K., 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=Environmental+and+genetic+influences+on+the+germination+of+Arabidopsis+thaliana+in+the+field.">Environmental and genetic influences on the germination of Arabidopsis thaliana in the field.</a> Evolution. 54 (4), 740-757 (2005).</li><li>Roach, D. A., Wulff, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+effects+in+plants.">Maternal effects in plants.</a> Ann Rev Ecol Syst. 18, 209-235 (1987).</li><li>Pace, B. A., Alexander, H. M., Emry, J. D., Mercer, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seed+fates+in+crop-wild+hybrid+sunflower:+crop+allele+and+maternal+effects.">Seed fates in crop-wild hybrid sunflower: crop allele and maternal effects.</a> Evol Appl. 8 (2), 121-132 (2015).</li><li>Alexander, H. M., Emry, D. J., Pace, B. A., Kost, M. A., Sparks, K. A., Mercer, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+maternal+effects+and+nuclear+genetic+composition+change+across+the+life+cycle+of+crop-wild+hybrids.">Roles of maternal effects and nuclear genetic composition change across the life cycle of crop-wild hybrids.</a> Am J Bot. 101 (7), 1176-1188 (2014).</li><li>Stewart, N. C., Matthew, J., Halfhill, D., Warwick, S. 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W., Rapset, M., Lienhard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tetrazolium+test+for+seed+viability.">The tetrazolium test for seed viability.</a> Mississippi Sta Uni Ag Exp Sta Techn Bull. 51, 1-63 (1962).</li><li>Association of Official Seed Analysts. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tetrazolium Testing Handbook. , (2010).</li><li>Alexander, H. M., Emry, D. J., Pace, B. A., Kost, M. A., Sparks, K. A., Mercer, K. 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The authors have nothing to disclose.

Qui vi presentiamo i metodi per l&#39;utilizzo di strisce di semi di sepoltura per osservare la germinazione del seme, dormienza e la mortalità delle scorte di sementi diverse in periodi di tempo preselezionato nel campo. I vantaggi di usare strisce anziché singoli pacchetti giacciono in (1) la velocità del nastro e la costruzione compartimento sul creazione di singoli pacchetti; e (2) la facilità e la velocità di rimozione comparti multipli in un unico movimento, senza il pericolo di omettere un pacchetto o la rim...

L&#39;obiettivo generale di questo metodo è quello di valutare in modo affidabile la sopravvivenza seme nel tempo in condizioni di campo. Banche del seme del terreno sono una riserva di dispersi, semi non germinati ancora vitali distribuiti sia sulla superficie del suolo, all&#39;interno lettiera di superficie o all&#39;interno del profilo del suolo, che possono persistere transitoriamente o per molti anni 1,2. Quando i metodi di sepoltura di semi simili a quelli presentati qui sono stati applicati ad uno studio di 17 anni con diverse decine di specie, semi vitali sono stati trovati in molte delle specie testate 3. Seed dormienza è un blocco di germinazione dei semi fino a quando la combinazione appropriata delle condizioni per la sopravvivenza piantina nascono 4. Rimanendo dormiente può permettere semi di sopravvivere condizioni difficili, come ad esempio basse temperature invernali, la limitazione di nutrienti, o siccità stagionale, fino a quando un trigger esterno per la dormienza-release consente per la germinazione. Trigger per dormienza-release possono variare da esposizione al freddo estesi, composti lasciati da fire, o attacco fisico sul cappotto di seme per abrasione o il contatto con gli acidi dello stomaco degli animali 5. Come spunti di germinazione possono essere generi o specie specifici e spesso il risultato di oltre la selezione naturale, disadattivi germinazione dei semi è quella che si verifica in un momento inopportuno, e può causare la mortalità semi o piantine o scarsa crescita delle piantine. Mentre dormienza è stato classificato in un certo numero di tipi basati sui meccanismi di dormienza rilascio (ad esempio, dormienza fisica, dormienza fisiologica), 6 seme dormienza rimane una delle meno argomenti inteso in biologia vegetale. Così, gli studi sul campo che consentono di valutare lo stato dei singoli semi o gruppi di semi in condizioni ecologiche rilevanti hanno una maggiore capacità esplicativa di quelli che semplicemente si basano su prove di germinazione standard in laboratorio. Lo sfruttamento delle caratteristiche del seme note in grado di fornire informazioni sui meccanismi di dormienza. Controllo di dormienza seme è complex, compreso il controllo genetico di fattori fisiologici e morfologici. Mentre una comprensione completa della larghezza di meccanismi dormienza è ancora stata chiarita, un modello generale è emersa, comportante un rapporto di feedback tra i due ormoni vegetali acido gibberellico (GA) e abscissico (ABA) 7. In questo modello generalizzato per semi con una componente fisiologica alla loro dormienza, GA serve come segnale di immissione dormienza, mentre ABA serve a mantenere lo stato dormiente. Effetti genetici materni, nonché l&#39;ambiente di crescita materno può influenzare dormienza e altri tratti di semi, come la dimensione, attraverso i tessuti maternamente generati ei segnali di sviluppo 8. Maternamente generato strutture esterne (o rivestimenti di semi) può mantenere la dormienza, a volte in combinazione con spunti fisiologici. Dal rivestimenti di semi di origine materna sono controllati dai geni della pianta madre, essi potrebbero non riflettere reale make-up genetico nucleare del seme. Noi ci abbiamoEd i acheni Helianthus annuus da una serie di ibrido crop-wild attraversa per prendere in giro questi materna vs. embrione effetti genetici sulle caratteristiche del seme 9,10. Così, disegni di studio che includono diverse specie, i tipi di croce, o genotipi possono raccogliere informazioni circa la ecologia e genetica di semi di dormienza, la germinazione e la sopravvivenza. Un importante esempio di come semi di germinazione e sopravvivenza fenotipi possono influenzare la dinamica delle popolazioni può essere visto in zone ibride colture-wild. Selezione durante la domesticazione delle piante coltivate elimina la maggior parte dormienza e riduce la capacità di un seme di sopravvivere al di fuori della stagione di crescita. Eppure flusso genico, o ibridazione, tra i tipi coltivate e selvatiche nelle zone ibride colture-wild possono reintrodurre alleli nel grano (o varianti genetiche) in una popolazione selvatica, con potenziali effetti sulle dinamiche della banca del seme. Ibridi tra parenti coltivate e selvatiche potenzialmente trovano in zone ibride colture-wild può possedereuna varietà di fenotipi dormienza intermedi, con pochi fenotipi attesi per sopravvivere condizioni di fuori della coltivazione (es mesi invernali) 11. Lo scopo di questo manoscritto è quello di mostrare come, utilizzando il metodo della striscia di sepoltura seme, siamo in grado di valutare la germinazione, dormienza, e la sopravvivenza di una vasta gamma di tipologie di semi in diversi periodi di tempo per indagare la loro variazione naturale in condizioni di campo. Nel nostro esempio, abbiamo impiegato semi di girasole da 15 tipi di ibridi incrociati colture-wild dal momento che siamo interessati ad effetti genetici materni ed embrione sulle caratteristiche del seme.

<table><tbody><tr><td>Small coin envelopes</td><td>Any</td><td></td><td></td></tr><tr><td>Large coin envelopes</td><td>Any</td><td></td><td></td></tr><tr><td>fine meshed polyester mosquito netting</td><td>Any</td><td></td><td></td></tr><tr><td>high-temperature glue gun</td><td>Any</td><td></td><td></td></tr><tr><td>high-temperature glue stick refills</td><td>Any</td><td></td><td></td></tr><tr><td>Industrial permenant markers</td><td>Any</td><td></td><td></td></tr><tr><td>plastic garden labels</td><td>Any</td><td></td><td></td></tr><tr><td>scissors</td><td>Any</td><td></td><td></td></tr><tr><td>Shovel</td><td>Any</td><td></td><td></td></tr><tr><td>Metal mesh hardward cloth</td><td>Any</td><td></td><td></td></tr><tr><td>Surveyor&#039;s flags, multiple colors</td><td>Any</td><td></td><td></td></tr><tr><td>Wet newspaper</td><td>Any</td><td></td><td></td></tr><tr><td>cooler</td><td>Any</td><td></td><td></td></tr><tr><td>blotter paper</td><td>Any</td><td></td><td></td></tr><tr><td>petri dishes</td><td>Any</td><td></td><td></td></tr><tr><td>Temp. controlled growth chamber</td><td>Any</td><td></td><td></td></tr><tr><td>razor blades</td><td>Any</td><td></td><td></td></tr><tr><td>Petri dishes</td><td>Any</td><td></td><td></td></tr><tr><td>Tetrazolium chloride</td><td>Any</td><td></td><td></td></tr><tr><td>water</td><td>Any</td><td></td><td></td></tr><tr><td>heat incubator</td><td>Any</td><td></td><td></td></tr></tbody></table>

reliable method for assessing seed germination, dormancy, and mortality under field conditions

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester fabric and glue can be folded and glued to construct a strip of compartments that house seeds and identifying information, while allowing contact with soil leachate, water, microorganisms, and ambient temperature. Strips may be constructed with a wide range of compartment numbers and sizes and allow the researcher to house a variety of genotypes within a single species, different species, or seeds that have experienced different treatments. As opposed to individual seed packets, strips are more easily retrieved as a unit. While replicate packets can be included within a strip, different strips can act as blocks or can be retrieved at different times for observation of seed behavior over time. We used a high temperature glue gun to delineate compartments and sealed the strips once the seed and tags identifying block and removal times were inserted. The seed strips were then buried in the field at the desired depth, with the location marked for later removal. Burrowing animal predators were effectively excluded by use of a covering of metal mesh hardware cloth on the soil surface. After the selected time interval for burial, strips were dug up and seeds were assessed for germination, dormancy and mortality. While clearly dead seeds can often be distinguished from ungerminated living ones by eye, dormant seeds were conclusively identified using a standard Tetrazolium chloride colorimetric test for seed viability.

Tipi incrociate con vario materna percentuale di parentela e delle colture allele (Tabella 1) differivano tra le date di rimozione in percentuale germinato, non germinato schiacciato, e semi di morti (Fig. 2 e 3). Utilizzando test TZ di semi non germinati, abbiamo trovato alcuni semi dormienti veramente al secondo rimozione (inizio primavera) (Tabella 2), mentre tutti i semi non germinati dalla terza rimozione (primavera...

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Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

Qui vi presentiamo un protocollo per la valutazione sopravvivenza dei semi, la germinazione e la dormienza in condizioni di campo che utilizzano sepolti, strisce sementi etichettate di test vitalità cloruro di tetrazolio (TZ).

Metodo affidabile per valutare la germinazione dei semi, dormienza, e mortalità in normali condizioni operative

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester ...

reliable-method-for-assessing-seed-germination-dormancy-mortality

Universidad de Cartagena UDEC

Research

JoVE Journal

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10.4K Views.  Ohio State University.  Qui vi presentiamo un protocollo per la valutazione sopravvivenza dei semi, la germinazione e la dormienza in condizioni di campo che utilizzano sepolti, strisce sementi etichettate di test vitalità cloruro di tetrazolio (TZ). 

Video: Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

Protocols for Robust Herbicide Resistance Testing in Different Weed Species

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented protocols, based on whole-plant bioassays performed in a greenhouse, can be readily adapted to a wide range of weed species and herbicides through appropriate variants. Seed samples from plants that survived a field herbicide treatment are collected and stored dry at low temperature until used. Germination methods differ according to weed species and seed dormancy type. Seedlings at similar growth stage are transplanted and maintained in the greenhouse under appropriate conditions until plants have reached the right growth stage for herbicide treatment. Accuracy is required to prepare the herbicide solution to avoid unverifiable mistakes. Other critical steps such as the application volume and spray speed are also evaluated. The advantages of this protocol, compared to others based on whole plant bioassays using one herbicide dose, are related to the higher reliability and the possibility of inferring the resistance level. Quicker and less expensive in vivo or in vitro diagnostic screening tests have been proposed (Petri dish bioassays, spectrophotometric tests), but they provide only qualitative information and their widespread use is hindered by the laborious set-up that some species may require. For routine resistance testing, the proposed whole plant bioassay can be applied at only one herbicide dose, so reducing the costs.

A robust and flexible approach to confirm herbicide resistance in weed populations is presented. This protocol allows the herbicide resistance levels to be inferred and applied to a wide range of weed species and herbicides with minor adaptations.

Protocolli per Robust erbicidi Test della resistenza in diversi Weed Specie

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented ...

Ricerca

Ambiente

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

Quantificazione di colonizzazione fungina, sporogenesi, e la produzione di micotossine Uso Bioassays Kernel

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

Immunologia e infezioni

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or complete loss of viability at different stages of development. Our lab has developed a method to assess viability in Drosophila from embryo to fully mature adult. The method relies on quantifying the number of progeny present at different stages during development, starting with hatched embryos. After embryos have been quantified, additional stages are counted, including L1/L2, pupae, and mature adults. After all stages have been examined, a statistical analysis such as the chi-square test is used to determine if there is a significant difference between the starting number of progeny (hatched embryos) and later stages culminating in the observed number of adults, thus rejecting or accepting the null hypothesis (that the number of hatched embryos will be equal to the number of larvae, pupae, and adults recorded throughout the stages of development). The primary advantage of this assay is its simplicity and accuracy, as it does not require an embryo rinse to transfer them to the food vial, avoiding losses from technical errors. Although the protocol described here does not directly examine L2/L3 larvae, additional steps can be added to account for these. Comparing the number of hatched embryos, L1, pupae, and adults can help determine if viability was compromised during the L2/L3 stages for further studies (the use of colored food helps with visual identification of larvae). Overall, this method can help Drosophila researchers and educators determine when viability is compromised during the fly life cycle. Routine assessment of stocks using this assay can prevent accumulation of secondary mutations that may affect the phenotype of the originally isolated mutant, especially if the original mutations affect fitness. For this reason, our lab maintains multiple copies of each of our Dm ime4 alleles and routinely checks the purity of each stock with this method in addition to other molecular analyses.&#160;

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

This protocol is designed to assess the viability of Drosophila at every developmental stage, from embryo to adult. The method can be used to determine and compare the viability of different genotypes or growth conditions.

Un metodo diretto e semplice per valutare la vitalità di Drosophila melanogasterda embrione ad adulto

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or ...

Biologia dello sviluppo

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a &#34;seed coat bedding&#34; assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

Un cappotto Seed Bedding Assay per esplorare geneticamente<em&gt; In Vitro</em&gt; Come la Germinazione Controlli endosperma del seme in<em&gt; Arabidopsis thaliana</em

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

Biologia

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

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

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

Metodo standardizzato per la sterilizzazione di High-throughput di semi di Arabidopsis

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

Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are thick-walled dispersal agents. Teliospores can remain dormant for decades. The dormancy is characterized by low metabolic rates, paused macromolecular biosynthesis and greatly reduced levels of respiration. Upon receiving required environmental signals, teliospores germinate to produce haploid cells, which can initiate new rounds of infection. Teliospore germination is characterized by the resumption of macromolecular biosynthesis, increased respiration and dramatic morphological changes. In order to precisely measure changes in cellular respiration during the early stages of germination, we have developed a simple protocol employing a Clark-type respirometer. The later stages of germination are distinguished by specific morphological changes, but germination is asynchronous. We developed a microdissection technique that enables us to collect teliospores at distinct germination stages.

Smut fungi cause many devastating agricultural diseases. They are dispersed as dormant teliospores that germinate in response to environmental cues. We outline two methods to investigate molecular changes during germination: measuring respiration increase to detect metabolic activation and assessing changing molecular events by isolating teliospores at distinct morphological stages.

Indagando Teliospore germinazione utilizzando analisi Microrespiration e Microdissection

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

Identificazione di nuovi regolatori della traspirazione vegetale mediante screening termico su larga scala in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, Rhizoctonia bataticola. In the literature, comprehensive and detailed step-by-step protocols on disease assays are sparse. This article provides complete details on the steps involved in setting up a blotting paper technique for quickly screening genotypes for resistance to DRR. The blotting paper technique is easy and less expensive. Another method, based on the sick pot approach, is a mimic of natural infection and can be applied to study the interacting components&#8212;plant, pathogen, and environment&#8212;involved in the disease triangle.
Moreover, in nature, DRR occurs mostly in rainfed chickpea cultivation areas, where soil moisture recedes as crop growth advances. Drought stress is known to predispose chickpea plants to DRR disease. Pathomorphological and molecular understanding of plant-pathogen interaction under drought stress can pave the way for the identification of elite DRR-resistant varieties from the chickpea germplasm pool. This article provides a stepwise methodology for the preparation of a sick pot and subsequent disease assay. Overall, the information presented herein will help researchers prepare R. bataticola fungal inoculum, maintain this pathogen, set up the blotting paper technique, prepare sick culture and sick pot, and assess pathogen infection in chickpea plants.

This study presents methodologies to study the pathomorphological and molecular mechanisms underlying chickpea&#8211;Rhizoctonia bataticola interaction. The blotting paper method is useful to rapidly study chickpea genotype responses, while the sick pot-based method can be used to simultaneously impose drought and R. bataticola infection and screen for tolerant genotypes.

Test della malattia del marciume delle radici secche nei ceci: una metodologia dettagliata

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, ...

High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental step required towards this end. Thus, it is paramount to establish high-throughput Arabidopsis seed surface sterilization methods to handle tens to hundreds of samples (e.g., transgenic lines, ecotypes, or mutants) at once. A seed surface sterilization method based on the efficient elimination of liquid in tubes with a homemade suction device constructed from a common vacuum pump is presented in this study. By dramatically reducing labor-intensive hands-on time with this method handling several hundreds of samples in one day is possible with little effort. Series time-course analyses further indicated a highly flexible time range of surface sterilization by maintaining high germination rates. This method could be easily adapted for surface sterilization of other kinds of small seeds with simple customization of the suction device according to the seed size, and the speed desired to eliminate the liquid.

A high-throughput protocol for the surface sterilization of Arabidopsisthaliana (Arabidopsis) seeds is provided, optimizing the liquid handling steps with a simple suction device constructed with a vacuum pump. Hundreds of seed samples can be surface-sterilized in one day.

Metodo ad alta produttività, robusto e altamente flessibile nel tempo per la sterilizzazione superficiale dei semi di Arabidopsis

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

Tecniche di microscopia per l'interpretazione della colonizzazione fungina nei tessuti delle piante micoeterotrofiche e della germinazione simbiotica dei semi

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...