Este trabajo fue apoyado por la Estación Experimental Agrícola de la Universidad de Cornell (CUAES) NYC-125433 Hatch Grant y Cornell subvención inicial concedida a OKV

Parte 1. Preparación de un medio sólido para el cultivo de plantas. Por lo general las plantas de la cultura en el concentrado Murasige y Skoog (MS) suplementado con 1% de sacarosa y 0,7% de agar. Contiene todos los ingredientes necesarios para mantener las plantas sanas. Para preparar 1 litro de MS, el pH 5,7 <ol><li> Añadir 2,15 g de MS polvo en 800 ml de agua en botella de 2,0 litros de 1,5 autoclavable. </li><li> Coloque una barra de agitación en la botella y disuelva el polvo MS agitando medio de una placa de agitación. </li><li> Mientras se agita, añadir gota a gota, KOH 1N para ajustar el pH del medio MS con 5,7. </li><li> Agregar 10 g de sacarosa, seguir revolviendo. </li><li> Añadir 7 g de agar *. </li><li> Ajustar el volumen final del medio preparado a 1000 ml y esterilizar en autoclave .* </li><li> Coloque el autoclave medio en una placa de agitación y enfriarlo mientras se agita, por lo que el agar no se precipitará en la parte inferior de la botella. Enfriamiento a medio y 60 º C. * </li><li> Vierta 100 mm placas de Petri. Mantenga las placas a 4 ° C. </li></ol> * Tip 1: No intente disolver el agar. Se solubilizan en autoclave. * Tip 2: Mantenga la barra de agitación en una botella durante el tratamiento en autoclave, ya que lo necesitará más adelante. * Consejo 3: Si usted puede mantener las manos en el frasco en un autoclave durante 10 s, significa que el medio se ha enfriado lo suficiente y se puede empezar a verter las placas. Parte 2. Material vegetal y condiciones de crecimiento. <ol><li> Esterilizar las semillas de Arabidopsis thaliana <ol><li> Colocar 100 mg de semillas en tubos de microcentrífuga eppendorf y agregar 1 ml de etanol al 70%. Mezclar bien e incubar durante 2 min. Girar las semillas hacia abajo, aspirar el sobrenadante. </li><li> Añadir 1 ml de solución de lejía al 1,8% * que contiene 0,1% de Tween-20. Mezcla durante 10 minutos. Decantar semillas y solución de cloro aspirado. </li><li> Este paso tiene que ser hecho en una campana de flujo laminar. Añadir 1 ml de agua estéril a las semillas, mezclar bien. De girar las semillas, se aspira el agua. Repita este paso 5 veces. </li></ol></li><li> Difundir las semillas en el preparado en el paso 1 placas (100 semillas / placa). </li><li> Mantenga placas sembradas durante 24-48 horas a 4 ° C en la oscuridad de la estratificación. </li><li> Crecer las plantas durante 14 días en la cámara de crecimiento de 8 h luz/16 h oscuridad fotoperíodo (con una densidad de flujo de fotones fotosintéticos de 250 mmol m -2 s -1) a 23 / ​​19 ° C de luz / oscuridad régimen de temperatura y 75% en relación humedad. </li></ol> * Tip 1: solución de cloro se hace-por dilución de un hogar de Clorox, que contiene 6% de hipoclorito de sodio y la adición de Tween-20 a una concentración final de 0,1%. Parte 3. El aislamiento de protoplastos a partir de plántulas de Arabidopsis. <ol><li> El segmento 2 g de 14 días de edad, las plántulas con una cuchilla de afeitar nueva en 15 ml de solución esterilizada por filtración, líneas de TV. Cortamos el material vegetal en estériles desechables platos de Petri. </li><li> La transferencia de los tejidos cortados en el vaso de 200 ml, 20 ml de solución enzimática esterilizada por filtración, se agita el vaso para que mezcla tejidos con la solución de la enzima, se cubre con parafina y papel de aluminio. </li><li> Agite tejidos de las plantas a 35 rpm en la oscuridad a temperatura ambiente durante 16-18 h. </li><li> Recoge los protoplastos liberados en 50 ml tubo Falcon por tamizado a través de ocho capas de la tela de queso, pre-húmedo en la solución W5. </li><li> Tamiz protoplastos a partir de la gasa una vez más por el lavado de la tela con 15 ml de solución W5. </li><li> Cuidadosamente protoplastos superposición con 10 ml de solución W5, no molestar el gradiente de azúcar; centrifugar durante siete minutos a 100 g. </li><li> Recoger 10 ml de protoplastos en la interfaz de la solución enzimática y W5 solución (Fig. 1B) y traslado a un nuevo tubo Falcon de 50 ml. </li><li> Agregar 15 ml de solución W5, centrifugar durante 5 min a 60 g. Eliminar el sobrenadante. </li><li> Lavado de protoplastos libre de solución de la enzima por resuspensión protoplastos en 15 ml de solución W5, centrifugar durante 5 minutos a 60 g </li><li> Retire los protoplastos sobrenadante, resuspender en pellets 1-3ml de solución W5. </li><li> Evaluar el rendimiento de protoplastos por conteo de células con un hemocitómetro. </li></ol> Parte 4. Reactivos: TVL: 0,3 M sorbitol, 50 mM de CaCl2 Filtro-esterilizar y almacenar a -20 ° C. Solución de la enzima: 0,5 M de sacarosa, 10 mM MES-KOH [pH 5.7], 20 mM CaCl 2, 40 mM KCl, 1% de celulasa (Onozuka R-10), el 1% Macerozyme (R10). Filtro de esterilización y el uso de recién. W5 Solución: 0,1% (w / v) de glucosa, 0,08% (w / v) de KCl, 0,9% (w / v) NaCl, 1,84% (w / v) CaCl 2, 2 mM MES-KOH pH 5,7. Filtro-esterilizar y almacenar a temperatura ambiente. Parte 5: Resultados del representante: Uso de las condiciones descritas en Arabidopsis cultura partes 1 y 2 hace plántulas sanas, adecuadas para el aislamiento de protoplastos (Fig. 1A). Protoplastos son purificados por gradientes de densidad de sacarosadiferentes centrifugación y se recoge en la interfaz de W5 y soluciones enzimáticas (Fig. 1B). El uso del procedimiento de protoplastos aislamiento descritas en la Parte 3 que por lo general la cosecha 5 al 10 octubre 6 de protoplastos intactos (Fig. 1C) de un gramo de plantas frescas. <img src="/files/ftp_upload/1149/Fig1.jpg" alt="Figura 1" /> Figura 1. El aislamiento de protoplastos a partir de plántulas de Arabidopsis. A, los tejidos de 14 días de plántulas de Arabidopsis se recogieron y se convierte en protoplastos mediante un procedimiento modificado de (Chen y Halkier, 2000). B, protoplastos fueron purificados por centrifugación en gradiente de densidad de sacarosa y se recoge en la interfaz de la solución enzimática y W5 uffer (flechas blancas). C, el microscopio de campo brillante de protoplastos. Microfotografías fueron obtenidos mediante una cámara CCD enfriado interfaz con el Axioscope Zeiss 2 más 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>

La patente provisional que describe el procedimiento para su uso en el análisis funcional de genes de plantas han sido presentados ante Cornell Centro para la Empresa de Tecnología y Comercialización (CCTEC), junio de 2008. Expediente n º 50341/015001: Vatamaniuk, en Aceptar y, Zhai, Z. Método rápido para análisis funcional de los genes de la planta.

Para asegurar el alto rendimiento de protoplastos intactos, es muy importante para la puesta en marcha de las plantas sanas. Use un filtro a esterilizar soluciones para el aislamiento de protoplastos. Recuerde que los protoplastos son frágiles. Por lo tanto, cuando usted está manejando protoplastos, no se mezclan, pipeta o vortex vigorosamente ya que les freno. En cambio, la mezcla protoplastos girando lentamente o grabar el tubo de centrífuga. El procedimiento descrito protoplastos intactos rendimiento que son viabl...

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<td>Murashige and Skoog Basal Salt Mixture (MS)</td>
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isolation of protoplasts from tissues of 14-day-old seedlings of arabidopsis thaliana

Los protoplastos son células vegetales que han tenido sus paredes celulares enzimáticamente eliminado. El aislamiento de protoplastos a partir de tejidos de plantas se registró por primera vez hace más de 40 años<sup&gt; 1</sup&gt; Y desde entonces ha sido adaptado para el estudio de una variedad de procesos celulares, tales como la localización subcelular de las proteínas, el aislamiento de los orgánulos intactos y de genes diana de la inactivación de doble cadena de ARN de interferencia (RNAi)<sup&gt; 2.5</sup&gt;. La mayoría de los protocolos de aislamiento de protoplastos utilizar tejidos de las hojas de Arabidopsis maduros (por ejemplo, 35 días de edad las plantas)<sup&gt; 2.4</sup&gt;. Hemos modificado los protocolos existentes mediante el empleo de 14 días de edad plántulas de Arabidopsis. En este procedimiento, un programa de 14 días de edad plántulas produjeron 5 10<sup&gt; 6</sup&gt; -10<sup&gt; 7</sup&gt; Protoplastos que permanecen intactos por lo menos 96 horas. El rendimiento de protoplastos de plantas es comparable con los preparativos de las hojas de Arabidopsis madura, pero en lugar de 35 a 36 días, el aislamiento de protoplastos se completa en 15 días. Esto permite disminuir el tiempo y el espacio de cámara de crecimiento que se requieren para el aislamiento de protoplastos de plantas adultas cuando se utilizan, y acelera los estudios posteriores que requieren protoplastos intactos.

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

Este video muestra un procedimiento para aislar protoplastos intactos los tejidos de 14 días de edad, las plántulas de Arabidopsis. Dado que los protoplastos aislados permanecen intactos por lo menos durante 96 horas y están aislados de plantas en vez de un mes de edad, las plantas adultas, este procedimiento acelera pruebas que requieren protoplastos intactos.

El aislamiento de protoplastos a partir de tejidos de 14 días de edad de las plántulas 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 vistas.  Cornell University. Este video muestra un procedimiento para aislar protoplastos intactos los tejidos de 14 días de edad, las plántulas de Arabidopsis. Dado que los protoplastos aislados permanecen intactos por lo menos durante 96 horas y están aislados de plantas en vez de un mes de edad, las plantas adultas, este procedimiento acelera pruebas que requieren protoplastos intactos.

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.

Aislamiento eficaz y rápida de los embriones en etapa temprana de<em&gt; Arabidopsis thaliana</em&gt; Semillas

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

Investigación

Biología

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.

La tinción histoquímica de<em&gt; Arabidopsis thaliana</em&gt; Celda Secundaria Elements Pared

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.

Análisis rápido de los fenotipos circadianos en Arabidopsis protoplastos transfectadas con un reloj luminiscentes Reportero

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.

Análisis del comportamiento de crecimiento de Arabidopsis thaliana en diferentes calidades de luz

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

Investigación espacio-temporal de la dinámica de ERL1 mediante imágenes confocales

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.

Autor destacado: Métodos basados en imágenes para estudiar los eventos de tráfico de membrana en células de linaje estomática  

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.

Métodos basados en imágenes para estudiar los eventos de tráfico de membrana en células de linaje estomática

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

Un método mejorado para medir los perfiles de hidratación del polen en 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.

Autor destacado: Un bioensayo de hidratación de polen in vivo de alta resolución, de un solo grano para 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 bioensayo de hidratación de polen in vivo de grano único de alta resolución para Arabidopsis thaliana

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