Dr. Stephen Alderman, il dottor David Leaf e Erin Macri Si ringraziano per l&#39;aiuto con la microscopia. Questa ricerca è stata finanziata attraverso un finanziamento della Specialità Colture Research Initiative NIFA, USDA SCRI-SREP di Grant Award No. 2.009-02.484. Briana Kinash, Kevin Kinloch, Megan Leonhard Joseph McCollum, Maria McSharry e Nicole Sallee fornito un&#39;eccellente assistenza tecnica e articolata discussione.

<ol>
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Gli autori dichiarano di non avere interessi finanziari in competizione.

La procedura descritta nel presente documento rappresenta una tecnica di primo passaggio per isolare potenziali degraders BDM dal suolo, ed è stato utilizzato con successo per isolare i funghi da BDM sepolte nel terreno per sette mesi. Funghi cresciuti quando reinoculated su materiale BDM fresca dello stesso tipo, indicando che i funghi isolate erano effettivamente colonizzatori, e che i film non erano inibire la crescita fungina. Isolamento di funghi e batteri che degradano plastica potenzialmente potrebbe portare al ...

La degradazione è stata storicamente considerata un attributo desiderabile di polimeri plastici, perché ripartizione accorcia durata del prodotto e la durata. Recentemente, la consapevolezza delle problematiche ambientali connesse ai rifiuti plastici nell&#39;ambiente naturale 1,2,3 ha fatto plastica biodegradabile una valida alternativa ai materiali tradizionali. Degradazione (definita come cambiamenti strutturali, la frammentazione e la riduzione del peso molecolare, l&#39;integrità e la forza 4,5) avviene attraverso una serie di eventi, tra cui i due processi abiotici (stress termico, foto-ossidazione, idrolisi, l&#39;erosione e lo stress meccanico), e degradazione biologica 6. Mentre processi abiotici possono cambiare la dimensione del frammento e caratteristiche di materie plastiche, microrganismi sono necessari per la loro mineralizzazione finale di acqua e anidride carbonica (in condizioni aerobiche) e / o metano (in condizioni anaerobiche). Una nicchia considerevole perplastiche biodegradabili esiste in agricoltura, dove pacciamatura plastica sono utilizzati per prevenire la crescita di erbacce, di trattenere l&#39;umidità del suolo e ad aumentare le temperature del terreno 7,8. Centinaia di migliaia di ettari nei soli Stati Uniti sono coperte di pacciamatura plastica 9, tra pacciamatura composti di plastica biodegradabile. A seguito di una stagione di crescita delle colture, le opzioni per lo smaltimento di pacciamatura biodegradabili (BDM) includono lo smaltimento in discarica, incenerimento per recupero di energia 10, la degradazione attraverso il compostaggio, o la degradazione nel suolo dopo la lavorazione del terreno 11. Di questi, il meno il destino di manodopera sta arando BDM nel terreno, ma senza degradazione efficiente e mineralizzazione nei mesi non colturali (generalmente in inverno), frammenti di plastica potrebbero rimanere e interferire con attrezzature agricole durante la lavorazione del terreno e semina primaverile, e persistono nell&#39;ambiente in cui essi influiscono in modo significativo la fauna selvatica, la vita vegetale, e microbiota 1,2,3,10. <p class = &quot;jove_content&quot;&gt; Anche se molti prodotti di plastica, tra cui film di pacciamatura agricola, si fregiano del marchio &quot;biodegradabile&quot; e &quot;compostabile&quot;, in pratica, la degradazione e la mineralizzazione possono essere troppo inefficienti e / o troppo incompleto per la decomposizione in-terreno sia un valida alternativa per lo smaltimento di questi prodotti. Ad esempio, polietileni oxo-biodegradabili ottenuti solo 12,4% di mineralizzazione dopo un anno di intemperie e tre mesi successivi in un 58 ° C compost, e meno della metà di quella quantità di mineralizzazione si è verificato quando la temperatura del compost era di 25 ° C 12. In inverno, le temperature del suolo in molte località sarebbero inferiori a nessuno di queste temperature, presumibilmente conseguente attività microbica ancora più basso e, di conseguenza, meno mineralizzazione. Oltre a rallentare la velocità di degradazione, l&#39;uso improprio del termine &quot;biodegradabile&quot;, ha portato a diffidare di questi prodotti da parte dei consumatori 13,14, compresi quelli del settore agricolo. La biodegradazione è la conversionedi polimeri a biossido di carbonio (e / o metano) e acqua 14 da microrganismi presenti in natura 4. Pertanto, la biodegradazione deve essere misurata chimicamente; l&#39;associazione fisica di microrganismi con un substrato non implica la degradazione microbica di tale materiale. Come parte di uno sforzo per esaminare l&#39;uso sostenibile della BDM in agricoltura, questo studio si è concentrato sulla scoperta di microrganismi autoctoni di suoli agricoli che colonizzano e degradano BDM disponibili in commercio. Metodi di prova standard sono stati pubblicati per la misurazione chimicamente la ripartizione di plastiche biodegradabili mediante abiotiche e biologiche 15,16,17. Tuttavia, questi metodi non affrontano degradazione di materie plastiche a singole specie microbiche, o forniscono metodi per il loro isolamento. La metodologia qui assomiglia più da vicino i metodi standard per la valutazione di plastica per la resistenza alla rottura microbica dopo inoculando i campioni con le spore fungine18,19. Quando formulazioni di materie plastiche sono noti e una fonte di materia prima è disponibile, plastica in polvere può essere sospesa in mezzi a base di agar e il degrado determinati dalla visualizzazione di zone di compensazione 13. Questo metodo è stato utilizzato in precedenza per identificare microrganismi che degradano i polimeri quali poliuretano 20, poli-(butilene succinato-co-adipato) 21, e di poli (acido lattico) 22. Un metodo simile comporta sospende plastica in polvere pura in terreno liquido in cui la plastica è l&#39;unica fonte di carbonio 20,23. Mentre questi metodi hanno il vantaggio di un sistema definito, essi mal mimare degradazione in situ di BDM. Prima, l&#39;area superficiale è distribuita in modo diverso perché BDM non sono dispersi in piccole particelle in tutta la matrice suolo, ma piuttosto, venduti e usati come film. In secondo luogo, la composizione chimica dei BDM è diverso da polimeri puri. BDM generalmente contengono additivi comeriempitivi, plastificanti, e coloranti, e questi additivi possono influenzare la crescita microbica e di conseguenza, il tasso di mineralizzazione. Per questa ragione, e perché la composizione di alcuni film commerciali in questo studio erano proprietarie, film plastico nella sua forma pronto per il campo è stato utilizzato per isolare funghi e batteri. Per semplicità, i metodi indicati sono descritte solo per i funghi, con le modifiche indicate dove appropriato per isolamenti batterici. In un recente studio 24, tre BDM disponibili in commercio e un film sperimentale sono stati utilizzati in siti agricoli in tre diverse regioni degli Stati Uniti per una stagione in crescita, e successivamente immesse in mesh (250 micron), borse e sepolti per un inverno nel suolo nello stesso sito. I 250 micron aperture di maglia consentono ife fungine di penetrare escludendo le radici e la maggior parte della fauna del suolo, e riducendo al minimo l&#39;invasione del terreno 25,26. Materiali di nylon prevenire il degrado sacchetto nel suolo. Seguendo scavo, fisolati ungal sono stati recuperati da pezzi BDM e valutati per la crescita in terreno minimo, senza una fonte di carbonio tranne l&#39;agar e di 5 cm x 5 cm quadrati di superficie disinfestare di nuovo, film di BDM non utilizzato che è stato pre-disinfestare. La maggior parte dei materiali plastici utilizzati come film non possono essere sterilizzati in autoclave senza perdita di integrità, la luce UV in modo da è stato utilizzato per uccidere tutte le cellule microbiche che risiedono sulle plastiche. ISO 846-19 raccomanda superficie disinfestante in etanolo 70% e successiva essiccazione, ma se si usa questo metodo, si deve garantire che nessun componente o additivo del film è influenzata negativamente dalla etanolo. Poiché BDM presumibilmente sono realizzati per resistere luce solare, UV è stata scelta come metodo di decontaminazione. Gli isolati che crescevano sulla BDM pezzi migliori rispetto a solo terreno minimo sono stati selezionati per ulteriori studi. Agar, un polisaccaride prodotto da alghe marine, è utilizzato per solidificare supporti microbiche perché non è utilizzato tipicamente da metabolicamente agricolo e non medicalmentemicrorganismi in grado, tuttavia, agar-enzimi idrolizzano sono stati isolati da batteri marini 27 e agar-idrolizzano anche batteri sono stati isolati dal suolo 28. Polimeri BDM e agar sono entrambi dovrebbero essere substrati rare per gli enzimi secreti da funghi del suolo, che non si sono evoluti in ambienti che contengono questi polimeri come potenziali fonti di nutrienti, ma entrambi i substrati sono presenti nel saggio biologico piastra qui descritto (punto 7). Funghi che utilizzano BDM ma non agar come fonte di carbonio può essere differenziata da funghi che utilizzano solo agar, confrontando crescita su agar-solidificato mezzo contenente i) senza aggiunta di fonte di carbonio tranne agar (controllo negativo), ii) BDM film (sperimentale) e iii) glucosio (controllo positivo). La crescita di tutti gli isolati è previsto in terreno minimo, più glucosio; funghi non derivanti da glucosio contenente piastre può non essere capace di una crescita del particolare mezzo minimo utilizzato nell&#39;esperimento. Potential BDM degraders dovrebbe crescere su agar-solidificato terreno minimo + film BDM meglio che crescono su agar-solidificato terreno minimo da solo. I funghi che crescono su piastre di terreno minimo sono agar-degraders o oligotrophs, e sono inoltre tenuti a crescere in agar associato con i film BDM in lastre saggi biologici, ma non sugli stessi film (a meno che non casualmente anche degradare polimeri BDM). Per eliminare la possibilità di vedere la crescita microbica a causa di utilizzo di agar e non di BDM, abbiamo seguito il nostro test iniziale per la colonizzazione BDM su piastre di agar con un saggio biologico in brodo definito (punto 9). Pezzi BDM rappresentato la fonte di carbonio noto solo nei tubi saggi biologici. Dopo lo screening iniziale, e su di rivivere le scorte glicerolo degli isolati, alcuni formati scarsa ma visibile miceli in terreno specifico liquido che contiene nessuna fonte di carbonio noto. Questi risultati suggeriscono che alcuni degli isolati acquisiti erano oligotrophs - organismi che crescono da scavenging molto piccole quantità di carbonio, azoto e altri nutrienti disciolti sia in ambiente acquoso o esistere come sostanze volatili nell&#39;aria 29,30,31. Identificazione delle specie tramite l&#39;analisi del DNA ribosomiale 18S sostenuto questo punto di vista, come molti degli isolati trovati generi fungini riferito precedentemente esporre oligotrofia 32. Oligotrophs, che sono comunemente saprofiti, richiedono una vasta gamma di capacità metaboliche di utilizzazione del substrato in una serie di ambienti 30. Quindi, non è sorprendente che gli stessi funghi abbiamo isolato dal BDM (presumibilmente richiedono particolari capacità enzimatiche) hanno dimostrato capacità oligotrofiche, e sono stati in grado di crescere su di contaminanti in tracce come oli per la pelle da impronte, polvere o tracce di sostanze volatili nell&#39;aria. A causa dell&#39;isolamento di oligotrophs, abbiamo concluso che la crescita su una superficie BDM soli, non potevano essere utilizzati per inferire ripartizione BDM. I metodi descritti nel presente documento riflettono i nostri sforzi per screen colonizzatori BDM nativi da suoli agricoli per bona fide ripartizione BDM.

<table border="1">
<tbody>
 <tr>
 <td>Reagent Name</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Potato Dextrose Agar</td>
 <td>Becton Dickinson</td>
 <td>8X05491</td>
 <td></td>
 </tr>
 <tr>
 <td >Agar</td>
 <td>Fisher</td>
 <td>BP 1423-2</td>
 <td></td>
 </tr>
 <tr>
 <td >Chloramphenicol</td>
 <td>Acros Organics</td>
 <td>200-287-4</td>
 <td></td>
 </tr>
 <tr>
 <td >Glutaraldehyde</td>
 <td>Electon Microscopy Sciences</td>
 <td>16216-10</td>
 <td>Toxic</td>
 </tr>
 <tr>
 <td >Molecular sieve</td>
 <td>Fisher </td>
 <td>M-8892</td>
 <td></td>
 </tr>
 <tr>
 <td >Ethanol</td>
 <td>Pharmco-Aaper</td>
 <td>E200</td>
 <td></td>
 </tr>
 <tr>
 <td >Contrex</td>
 <td>Decon Labs, Inc.</td>
 <td>5204</td>
 <td></td>
 </tr>
 <tr>
 <td >Parafilm M</td>
 <td>Pechiney Plastic Packaging</td>
 <td>S37440</td>
 <td></td>
 </tr>
 <tr>
 <td >Mineral salts for buffers and media</td>
 <td>Fisher</td>
 <td>Various</td>
 <td>Various vendors sell these reagents</td>
 </tr>
 </tbody>
</table>

isolation of native soil microorganisms with potential for breaking down biodegradable plastic mulch films used in agriculture

I funghi nativi per i suoli agricoli che colonizzarono pacciamatura (BDM) film biodegradabili in commercio sono stati isolati e valutati per la capacità di degradare plastica. Tipicamente, quando le formulazioni di materie plastiche sono noti e una fonte di materia prima è disponibile, plastica in polvere può essere sospesa in mezzi a base di agar e il degrado determinati dalla visualizzazione di zone di compensazione. Tuttavia, questo approccio imita scarsamente degradazione in situ di BDM. Prima, BDM non vengono disperse come piccole particelle in tutta la matrice suolo. In secondo luogo, BDM non vengono commercializzate come polimeri puri, ma piuttosto come i film che contengono additivi (ad esempio riempitivi, plastificanti e coloranti) che possono influenzare la crescita microbica. Le procedure descritte nel presente documento sono stati utilizzati per gli isolati acquisiti da film pacciamatura condizioni di interramento. Isolati fungini acquisiti da BDM scavati sono stati testati individualmente per la crescita su pezzi di nuove, BDM disinfestati cui cima di un terreno specifico che contiene nessuna fonte di carbonio eXCEPT agar. Gli isolati che crescevano sulla BDM sono stati ulteriormente testati in mezzo liquido in cui BDM erano l&#39;unica fonte di carbonio aggiunto. Dopo circa dieci settimane, la colonizzazione fungina e degrado BDM sono stati valutati mediante microscopia elettronica a scansione. Isolati sono stati identificati mediante analisi delle sequenze geniche di RNA ribosomiale. Questo rapporto descrive i metodi per l&#39;isolamento da funghi, ma i batteri sono stati isolati anche con questi metodi da parte dei media sostitutivi adeguati per i batteri. La nostra metodologia dovrebbe rivelarsi utile per gli studi indagano ripartizione dei film plastici intatti o prodotti per i quali materie prime plastiche sono o sconosciuti o non disponibile. Tuttavia il nostro approccio non fornisce un metodo quantitativo per confrontare i tassi di degrado BDM.

In un recente studio 24, quattro repliche ciascuno dei tre BDM disponibili in commercio etichettati &quot;biodegradabili&quot;, più un film sperimentale e un controllo di plastica convenzionale, sono stati collocati sopra suolo come concime per la produzione di pomodoro nella primavera del 2010 a Mount Vernon, WA, Knoxville, TN, e Lubbock, TX. Nell&#39;autunno del 2010, le piazze cinematografiche BDM sono stati tagliati da ogni intemperie pacciamatura in quattro appezzamenti replicare e suolo natio è stato ...

Watch this Scientific Journal Video about Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture at JoVE.com

Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture

Pellicole in plastica etichettati &quot;biodegradabili&quot; sono disponibili in commercio per uso agricolo come pacciamatura. Lavorazione del terreno rappresenta un metodo di smaltimento attraente, ma la degradazione in condizioni di campo è poco conosciuta. Lo scopo di questo studio è stato quello di sviluppare metodi per isolare funghi del suolo nativo e batteri che colonizzano i film di pacciamatura plastica dopo il campo di inumazione.

Isolamento dei nativi microrganismi del suolo con un potenziale di Breaking Down biodegradabili di pacciamatura film plastici utilizzati in agricoltura

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to ...

isolation-native-soil-microorganisms-with-potential-for-breaking-down

Research

JoVE Journal

Biology

30.4K Views.  Western Washington University. Pellicole in plastica etichettati "biodegradabili" sono disponibili in commercio per uso agricolo come pacciamatura. Lavorazione del terreno rappresenta un metodo di smaltimento attraente, ma la degradazione in condizioni di campo è poco conosciuta. Lo scopo di questo studio è stato quello di sviluppare metodi per isolare funghi del suolo nativo e batteri che colonizzano i film di pacciamatura plastica dopo il campo di inumazione.

Video: Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

Incorporare plastica e Sezionamento embrioni di Xenopus laevis

Ricerca

Biologia

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

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

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

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

Isolamento di protoplasti da tessuti di 14-giorni di età piantine di Arabidopsis thaliana</em

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

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) plasmid. A. rhizogenes can cause hairy root formation on plant tissues and form composite plants after transformation. On these composite plants, some of the regenerated roots are transgenic, carrying the wild type T-DNA and the engineered binary vector; while the shoots are still non-transgenic, serving to provide energy and growth support. These hairy root composite plants will not produce transgenic seeds, but there are a number of important features that make these composite plants very useful in plant research. First, with a broad host range,A. rhizogenes can transform many plant species, especially dicots, allowing genetic engineering in a variety of species. Second, A. rhizogenes infect tissues and explants directly; no tissue cultures prior to transformation is necessary to obtain composite plants, making them ideal for transforming recalcitrant plant species. Moreover, transgenic root tissues can be generated in a matter of weeks. For Medicago truncatula, we can obtain transgenic roots in as short as three weeks, faster than normal floral dip Arabidopsis transformation. Overall, the hairy root composite plant technology is a versatile and useful tool to study gene functions and root related-phenotypes. Here we demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species M. truncatula.

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

We demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species Medicago truncatula.

Generazione di piante Composite in Medicago truncatula Per saggi nodulazione

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

Legatura altamente efficiente delle piccole molecole di RNA per microRNA Quantificazione mediante sequenziamento High-Throughput

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

Visualizzazione e quantificazione del potenziale di differenziazione delle cellule mesenchimali adipogenico con un Marker specifico lignaggio

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

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

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

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

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

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

Repeatable Stair-step Assay to Access the Allelopathic Potential of Weedy Rice (Oryza sativa ssp.)

Weed competition contributes significantly to yield losses in cropping systems worldwide. The evolution of resistance in many weed species to continuously applied herbicides has presented the need for additional management methods. Allelopathy is a physiological process that some plant species possess that provide the plant with an advantage over its neighbors. Allelopathic crop varieties would be equipped with the ability to suppress the growth of surrounding competitors, thus reducing potential yield loss due to weed interference. This paper focuses on the construction and operation of a stair-step assay used for the screening of the allelopathic potential of a donor species (Oryza sativa) against a receiver weed species (Echinochloa crus-galli) in a greenhouse setting. The structure described in this paper serves as a stand for the plant samples and incorporates a timed watering system for the accumulation and distribution of allelochemicals. Allelochemicals produced by the plant roots are allowed to flow downward through a series of four pots separately into a collection tank and recycled back to the top plant through electric pumps. This method of screening provides an avenue for the allelochemicals from the donor plant to reach receiver plants without any resource competition, thus allowing quantitative measurement of the allelopathic potential of the selected donor plant. The allelopathic potential is measurable through the height reduction of the receiver plants. Preliminary screening data for the effectiveness of this method demonstrated height reduction in the receiver species, barnyardgrass (E. crus-galli), and thus the presence of allelopathic residues from the donor plant, weedy rice (Oryza sativa).

Repeatable Stair-step Assay to Access the Allelopathic Potential of Weedy Rice Oryza sativa ssp.

Allelopathy has shown promise as a useful supplemental weed control strategy in cropping systems. To determine the allelopathic potential of a desired plant specimen, a stair-step screening method is provided.

Analisi in fase di stadi ripetibile per accedere al potenziale allelopatico del riso erbaceo (Oryza sativa ssp.)

Weed competition contributes significantly to yield losses in cropping systems worldwide. The evolution of resistance in many weed species to continuously ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

Microsezione laser-cattura RNA-Sequenziamento per l'analisi dell'espressione genica spaziale e temporale specifica del tessuto nelle piante

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

Isolamento degli epatociti primari di ratto con controllo della perfusione multiparametrica

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

Applicazione del movimento passivo della testa per generare accelerazioni definite alle teste dei roditori

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...