Questo lavoro è stato sostenuto dal Experiment Station Cornell University agricolo (CUAES) NYC-125433 Hatch Grant e Cornell Start-Up sovvenzione concessa a OKV

Parte 1. Preparazione del terreno solido per le piante in crescita. Noi di solito le piante sulla cultura concentrato Murasige e Skoog (MS) media integrato con 1% di saccarosio e lo 0,7% agar. Esso contiene tutti gli ingredienti necessari per mantenere le piante sane. Per preparare 1L di MS, media pH 5,7 <ol><li> Aggiungere 2,15 g di MS polvere in 800 ml di acqua in bottiglia da 1,5 2.0L autoclavabile. </li><li> Posizionare un bar mescolando nella bottiglia e si dissolvono in polvere MS agitando medio su un piatto mescolando. </li><li> Mescolando, aggiungere, goccia a goccia, KOH 1N per aggiustare il pH medio di MS a 5,7. </li><li> Aggiungere 10 g di saccarosio, continuare a mescolare. </li><li> Aggiungono 7 g di agar *. </li><li> Regolare il volume finale del terreno preparato a 1000 ml e sterilizzare in autoclave .* </li><li> Luogo autoclave medio su un piatto mescolando e raffreddarlo mescolando, in modo che agar non precipitare sul fondo della bottiglia. Raffreddare medio ~ 60 ° C. * </li><li> Versare 100 millimetri piastre Petri. Tenere le piastre a 4 ° C. </li></ol> * Suggerimento 1: Non tentare di sciogliere agar. Si solubilizzano durante la sterilizzazione in autoclave. * Suggerimento 2: Tenere la barra mescolando in una bottiglia durante la sterilizzazione, ne avrete bisogno in seguito. * Suggerimento 3: Se potete tenervi le mani sulla bottiglia in autoclave per 10 s, significa che il mezzo si raffreddi e si può cominciare a diffondere piatti. Parte 2. Materiale vegetale e condizioni di crescita. <ol><li> Sterilizzare i semi di Arabidopsis thaliana <ol><li> Mettere 100 mg di semi in provette da microcentrifuga eppendorf e aggiungere 1 ml di etanolo al 70%. Mescolare bene e incubare per 2 minuti. Spin semi giù, aspirare il sopranatante. </li><li> Aggiungere 1 ml di soluzione di candeggina al 1,8% * contenente 0,1% di Tween-20. Mescolare per 10 min. Spin down semi e soluzione di candeggina aspirare. </li><li> Questo passo deve essere fatto in una cappa a flusso laminare. Aggiungere 1 ml di acqua sterile per i semi, mescolare bene. Spin down semi, aspirare l&#39;acqua. Ripetere questa operazione per 5 volte. </li></ol></li><li> Diffondere i semi sul preparato nella fase 1 piastre (100 semi / piastra). </li><li> Conservare le piastre seminate per 24-48 ore a 4 ° C al buio per la stratificazione. </li><li> Coltivare piante per 14 giorni nella camera di crescita con 8 h light/16 h scuro fotoperiodo (con una densità di fotosintesi flusso di fotoni di 250 mmol m -2 s -1) a 23 / ​​19 ° C, luce / buio regime di temperatura e il 75% rispetto umidità. </li></ol> * Suggerimento 1: soluzione di Bleach è composto da una famiglia di diluizione Clorox, contenente ipoclorito di sodio al 6% e l&#39;aggiunta di Tween-20 a una concentrazione finale di 0,1%. Parte 3. Isolamento di protoplasti da piantine di Arabidopsis. <ol><li> Fetta 2 g di 14-giorno-vecchio piantine con una lama di rasoio fresco in 15 ml di soluzione sterilizzata per filtrazione, TVL. Noi tagliare materiale vegetale in piatti monouso sterili Petri. </li><li> Trasferimento tessuti tagliati in 200 ml bicchiere, aggiungere 20 ml di soluzione enzimatica, sterilizzata per filtrazione, agitare il bicchiere per far mescolare tessuti con soluzione enzimatica, coprire con parafilm e foglio di alluminio. </li><li> Agitare tessuti vegetali a 35 giri al buio a temperatura ambiente per 16-18 h. </li><li> Raccogliere la protoplasti rilasciata nel tubo da 50 ml Falcon mediante setacciatura a 8 strati di telo, pre-umido in W5 soluzione. </li><li> Protoplasti setaccio dal formaggio panno ancora una volta con il lavaggio il panno con 15 ml di soluzione W5. </li><li> Attentamente protoplasti sovrapposizione con 10 ml di soluzione W5, non disturbare il gradiente di zucchero; centrifugare per 7 minuti a 100 g. </li><li> Raccogliere 10 ml di protoplasti a livello di interfaccia di soluzione enzimatica e W5 Soluzione (Fig. 1B) e trasferirli in un nuovo tubo da 50 ml Falcon. </li><li> Aggiungere 15 ml di soluzione W5, centrifugare per 5 min a 60 g. Rimuovere il surnatante. </li><li> Lavare protoplasti privi di soluzione enzimatica da risospendere protoplasti in 15ml di soluzione W5, centrifugare per 5 min a 60 g </li><li> Rimuovere il surnatante, risospendere protoplasti pellet in 1-3ml W5 soluzione. </li><li> Valutare resa protoplasti da cellule contando con un emocitometro. </li></ol> Parte 4. Reagenti: TVL: 0,3 M sorbitolo; 50 mM CaCl 2 Filtro-sterilizzare e conservare a -20 ° C. Enzima Soluzione: 0,5 M saccarosio, 10 mM MES-KOH [pH 5,7], 20 mM CaCl 2, 40 mM KCl, 1% Cellulase (Onozuka R-10), 1% Macerozyme (R10). Filtro-sterilizzare e appena uso. W5 Soluzione: 0,1% (w / v) di glucosio, 0,08% (w / v) KCl, 0,9% (w / v) di NaCl, 1,84% (w / v) CaCl 2, 2 mM MES-KOH pH 5.7. Filtro-sterilizzare e conservare a temperatura ambiente. Parte 5: Risultati Rappresentante: Utilizzando condizioni di coltura Arabidopsis descritti nelle parti 1 e 2 piantine rende sani, adatti per isolare protoplasti (Fig. 1A). Protoplasti sono purificati dai gradienti di densità di saccarosioENT centrifugazione e raccolti a livello di interfaccia di W5 e soluzioni di enzimi (Fig. 1B). Utilizzando l&#39;isolamento di protoplasti procedura descritta nella parte 3 abbiamo raccolto tipicamente 5-10 ottobre 6 di protoplasti intatti (Fig. 1C) da un grammo di piantine fresche. <img src="/files/ftp_upload/1149/Fig1.jpg" alt="Figura 1" /> Figura 1. Isolamento di protoplasti da piantine di Arabidopsis. A, tessuti dal 14-giorni di età piantine di Arabidopsis sono stati raccolti e convertiti in protoplasti con una procedura modificata (Chen e Halkier, 2000). B, protoplasti sono stati purificati mediante centrifugazione gradiente di densità di saccarosio e raccolti a livello di interfaccia della soluzione enzimatica e W5 Uffer (frecce bianche). C, luminoso-campo della microscopia protoplasti. Microfotografie sono stati raccolti utilizzando una camera CCD raffreddata interfacciato con il Axioscope Zeiss 2 plus microscopio.

<ol>
	<li>Cocking, E. 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+Method+for+the+Isolation+of+Plant+Protoplasts+and+Vacuoles.">A Method for the Isolation of Plant Protoplasts and Vacuoles.</a> Nature. 187, 962-963 (1960).</li><li>Chen, S., Halkier, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+glucosinolate+uptake+by+leaf+protoplasts+of+Brassica+napus.">Characterization of glucosinolate uptake by leaf protoplasts of Brassica napus.</a> J Biol Chem. 275, 22955-22960 (2000).</li><li>Robert, S., Zouhar, J., Carter, C., Raikhel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+intact+vacuoles+from+Arabidopsis+rosette+leaf-derived+protoplasts.">Isolation of intact vacuoles from Arabidopsis rosette leaf-derived protoplasts.</a> Nat. Protocols. 2, 259-262 (2007).</li><li>Yoo, S. D., Cho, Y. H., Sheen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+mesophyll+protoplasts:+a+versatile+cell+system+for+transient+gene+expression+analysis.">Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis.</a> Nat Protoc. 2, 1565-1572 (2007).</li><li>Zhai, Z., Sooksa-nguan, T., Vatamaniuk, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+RNAi+as+a+reverse+genetic+approach+for+gene+functional+analysis+in+protoplasts.">Establishing RNAi as a reverse genetic approach for gene functional analysis in protoplasts.</a> Plant Phys. 149, 642-652 (2009).</li></ol>

Il brevetto provvisorio che descrive la procedura per il suo utilizzo in analisi funzionali di geni della pianta è stato depositato presso la Cornell Center For Enterprise Technology e commercializzazione (CCTEC), giugno 2008. Docket Nessun 50341/015001: Vatamaniuk, OK e Zhai, Metodo Z. per una rapida analisi funzionali di geni della pianta.

Per garantire l&#39;alto rendimento di protoplasti intatti, è molto importante per start-up con piante sane. Utilizzare un filtro-sterilizzare le soluzioni per l&#39;isolamento protoplasti. Ricordate che protoplasti sono fragili. Pertanto, quando si sono movimentazione protoplasti, non si mescolano, o vortice pipettare vigorosamente perché li freni. Invece, mescolare protoplasti lentamente ruotando o con nastro adesivo il tubo di centrifuga. La procedura descritta protoplasti resa intatta che sono vitali per almeno 96...

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isolation of protoplasts from tissues of 14-day-old seedlings of arabidopsis thaliana

Protoplasti sono cellule vegetali che hanno avuto le loro pareti cellulari enzimaticamente rimosso. Isolamento di protoplasti dai tessuti vegetali diversi per la prima volta segnalato più di 40 anni fa<sup&gt; 1</sup&gt; E da allora è stato adattato per studiare una serie di processi cellulari, come la localizzazione subcellulare di proteine, l&#39;isolamento di organelli intatti e mirata gene-inattivazione con un doppio filamento di RNA interference (RNAi)<sup&gt; 2-5</sup&gt;. La maggior parte dei protocolli di isolamento del protoplasti utilizzare tessuti foglia di Arabidopsis maturi (per esempio 35 giorni di età delle piante)<sup&gt; 2-4</sup&gt;. Abbiamo modificato i protocolli esistenti con l&#39;impiego di 14 giorni di età piantine di Arabidopsis. In questa procedura, un grammo di 14-giorno-vecchie piantine prodotto 5 10<sup&gt; 6</sup&gt; -10<sup&gt; 7</sup&gt; Protoplasti che rimangono intatte, almeno 96 ore. La resa di protoplasti da piantine è paragonabile con i preparati da foglie di Arabidopsis maturo, ma invece di 35-36 giorni, l&#39;isolamento di protoplasti si completa in 15 giorni. Ciò consente di diminuire il tempo e lo spazio camera di crescita che sono necessari per isolare protoplasti quando le piante mature sono utilizzati, e accelera gli studi a valle che richiedono protoplasti intatti.

Watch this Scientific Journal Video about Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana at JoVE.com

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

Questo video mostra una procedura per isolare protoplasti intatti da tessuti di 14-giorno-vecchie piantine di Arabidopsis. Dato che il protoplasti isolati rimangono intatte per almeno 96 ore e sono isolati da piantine invece di piante mature di un mese-vecchia, questa procedura accelera i test che richiedono protoplasti intatti.

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 ...

isolation-protoplasts-from-tissues-14-day-old-seedlings-arabidopsis

Research

JoVE Journal

Biology

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

35.7K Views.  Cornell University. Questo video mostra una procedura per isolare protoplasti intatti da tessuti di 14-giorno-vecchie piantine di Arabidopsis. Dato che il protoplasti isolati rimangono intatte per almeno 96 ore e sono isolati da piantine invece di piante mature di un mese-vecchia, questa procedura accelera i test che richiedono protoplasti intatti.

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

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

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

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

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

Isolamento efficiente e rapida di embrioni nelle prime fasi di<em&gt; Arabidopsis thaliana</em&gt; Semi

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

Ricerca

Biologia

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

Colorazione istochimica di<em&gt; Arabidopsis thaliana</em&gt; Cellula secondaria Elementi da parete

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

Analisi Rapida di fenotipi circadiani in Arabidopsis protoplasti transfettate con un luminescenti Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light conditions, which are preferably variable in quantity and quality so that studies under different setups can be conducted. These requirements are met by climatic chambers featuring light emitting diodes (LED) lights, which can &#8211; in contrast to fluorescent lights &#8211; be set to different wavelengths. LEDs are energy conserving and emit virtually no heat even at light intensities, which often constitutes a problem with other light sources. The presented protocol provides a step-by-step guidance of how to program a climatic chamber equipped with variable LED lights as well as describing several approaches for in depth analysis of growth phenotypes. Depending on the experimental set-up various characteristics of the growing plants can be observed and analyzed. Here we describe how to determine fresh weight, leaf area, photosynthetic activity, and stomatal density. We demonstrate that in order to obtain reliable data and draw valid conclusions it is mandatory to use a sufficient number of individuals for statistical evaluation. Taking too few plants for this kind of analysis results in high statistical errors and consequently in less clear interpretations of the data.

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Here, we present a protocol for studying plant growth behavior and especially phenotypes in a reproducible manner. We show how to provide variable and at the same time stable light conditions. Proper analyses depend on sufficient sample numbers and valid statistical evaluations.

Analisi del comportamento di crescita di Arabidopsis thaliana in diverse qualità di luce

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light ...

Indagine spaziotemporale della dinamica ERL1 tramite imaging confocale

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

Autore in primo piano: Metodi basati su immagini per studiare gli eventi di traffico di membrana nelle cellule del lignaggio stomatico  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

Metodi basati su immagini per studiare eventi di traffico di membrana in cellule di lignaggio stomatico

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...

Un metodo migliorato per misurare i profili di idratazione del polline in Arabidopsis thaliana

A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is established between the interacting partners. Studies across a range of species have revealed that a series of molecular checkpoints regulate the pollen-stigma interaction to ensure that only compatible, generally intraspecific pollen is successful in effecting fertilization. In species that possess a 'dry stigma', such as the model plant Arabidopsis thaliana, the first post-pollination, prezygotic compatibility checkpoint is the establishment of pollen hydration. 
This phase of pollination is tightly regulated, whereby signals from the pollen grain elicit the release of water from the stigma, thus permitting pollen hydration. The ability to accurately measure and track pollen hydration over time is key to the design of experiments directed at understanding the regulation of this critical step in reproduction. Published protocols frequently utilize flowers that have been excised from the parent plant, maintained on liquid or solid media, and bulk pollinated. 
This paper describes a noninvasive, in vivo pollination bioassay that permits minute-by-minute hydration tracking of individual A. thaliana pollen grains at high resolution. The assay is highly reproducible, able to detect very subtle variations of pollen hydration profiles, and thus is suitable for the analysis of mutants that affect pathways regulating pollination. Although the protocol is lengthier than those described for bulk pollinations, the precision and reproducibility it provides, along with its in vivo nature, make it ideal for the detailed dissection of pollination phenotypes.

Autore in primo piano: Un saggio biologico sull'idratazione del polline in vivo ad alta risoluzione, a grana singola, per Arabidopsis thaliana  

An improved method to measure pollen hydration profiles in Arabidopsis thaliana is described here. The new method offers higher resolution, is noninvasive, and is highly reproducible. The protocol represents a new tool for a finer dissection of the processes that regulate the early stages of pollination.

Un saggio biologico di idratazione del polline in vivo ad alta risoluzione, a granulo, singolo grano per Arabidopsis thaliana

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is ...