Name: histochemical staining of arabidopsis thaliana secondary cell wall elements
Uploaded: 2014-05-13T23:10:00
Description: Watch this Scientific Journal Video about Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements at JoVE.com

Vi er taknemmelige for Sabin Russell til redigering assistance. Dette arbejde var en del af DOE Joint BioEnergy Institute (http://www.jbei.org), støttet af det amerikanske Department of Energy, Office of Science, Kontoret for Biologiske og Miljøforskning gennem kontrakt DE-AC02-05CH11231 mellem Lawrence Berkeley National Laboratory og US Department of Energy.

1.. Stem Embedding <ol><li> Lav en 7% agaroseopløsning i vand (7 g elektroforese-agarose i 100 ml destilleret vand). Opløses agarosen ved autoklavering i 20 minutter eller ved mikrobølgeovn i 20 minutter ved laveste intensitet (f.eks 10% intensiteten af en 1.250 watt mikrobølgeovn). </li><li> Forbered en hjemmelavet støbeform for indlejring stænglerne ved hjælp af plast hætteglas. <ol><li> Ved hjælp af et barberblad, afbrød den koniske bund af en 2 ml skruelåg mikrocentrifugerør (del A). Med en ka...

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M., Wood, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Congo+red-polysaccharide+interactions+in+enumeration+and+characterization+of+cellulolytic+bacteria+from+the+bovine+rumen.">Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen.</a> Applied and environmental microbiology. 43, 777-780 (1982).</li><li>Yeung, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+beginner's+guide+to+the+study+of+plant+structure.">A beginner's guide to the study of plant structure.</a> Proceedings of the 19th Workshop/Conference of the Association for Biology Laboratory Education (ABLE. 19, 365-36 (1998).</li><li>Turner, S. R., Somerville, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collapsed+xylem+phenotype+of+Arabidopsis+identifies+mutants+deficient+in+cellulose+deposition+in+the+secondary+cell+wall).">Collapsed xylem phenotype of Arabidopsis identifies mutants deficient in cellulose deposition in the secondary cell wall).</a> The Plant Cell Online. 9, 689-701 (1997).</li><li>Iiyama, K., Pant, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+the+M&#228;ule+colour+reaction.+Introduction+of+methylated+syringyl+nuclei+into+softwood+lignin.">The mechanism of the M&#228;ule colour reaction. Introduction of methylated syringyl nuclei into softwood lignin.</a> Wood Sci. Technol. 22, 167-175 (1988).</li><li>Yang, F., 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=Engineering+secondary+cell+wall+deposition+in+plants.">Engineering secondary cell wall deposition in plants.</a> Plant biotechnology journal. 11, 325-335 (2013).</li><li>Zhong, R., Ripperger, A., Ye, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+deposition+of+lignin+in+the+pith+of+stems+of+two+Arabidopsis+mutants.">Ectopic deposition of lignin in the pith of stems of two Arabidopsis mutants.</a> Plant physiology. 123, 59-70 (2000).</li><li>Chapple, C. C., Vogt, T., Ellis, B. E., Somerville, C. 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A. thaliana stamceller sektioner er almindeligt anvendt til at undersøge organiseringen af cellerne i den sekundære cellevæg og kvalitativt undersøge forskellene mellem vildtype og transgene planter. De almindeligt anvendte teknikker til skæring er prøverne direkte hånd opskæring; eller når prøver er indlejret i agarose eller fiksativ, kan sektionering gøres med en vibratome eller mikrotom. I modsætning til hånd skæring, tillader de to sidste mindske risikoen for at producere unøjagtige forskelle...

Plantecellevæggen rummer et væld af oplysninger i sine forskellige komponenter: lignin, cellulose, hemicellulose (xylan, glucuronoxylan, xyloglucan, arabinoxylan, blandet kobling glucan eller glucomannan), og pektin. Histologiske teknikker giver vigtige visuelle referencer i at studere forskellene inden for de sekundære cellevægge på organisatoriske og cellulære niveauer. Forskellige histologiske teknikker blev udviklet og kan findes i litteraturen, men disse teknikker kan være udfordrende og tidskrævende for begyndere, da meget detaljerede protokoller med simple visuelle instruktioner er sjældent om nogensinde tilgængelige. Målet med denne undersøgelse er at give enkle retningslinjer for histologiske farvningsteknikker at opnå billeder af høj kvalitet. Sektionering stilken væv er det første skridt i visualisering af cellevægge og celle figurer. Selvom hånd-cut sektioner er billig og tage mindre tid til at forberede, brug af en vibratome giver konsistens oggiver billeder i høj kvalitet. Ved hjælp af en vibratome tillader at producere en bedre datakvalitet ved at generere endda sektioner med samme tykkelse, som hjælper med at producere skarpe billeder og reducerer risikoen for at producere unøjagtige forskelle mellem prøver, der ville simpelthen forårsaget af en dårlig prøveforberedelse. Brug harpiks til at fastsætte friske prøver kan være en udfordring for begyndere og kan stadig være tidskrævende selv for eksperter, når en analyse skal gøres hurtigt. Desuden bliver det muligt at måle enhver biologisk aktivitet med prøven, når den er indlejret i en harpiks. En simpel teknik, der anvender agarose og en hjemmelavet formen er nyttig for indlejring stamceller væv, og det kan også anvendes i andre applikationer, der kræver dissektion af det bløde væv. Sammenlignet med indlejring prøver i harpiks, har denne fremgangsmåde den fordel, at holde væv i live og reducere prøve manipulation. Sektionering væv via en vibratome er meget præcis og genererer homogenskellige sektioner, som, afhængigt af formålet med undersøgelsen, så kan anvendes med flere forskellige farvningsteknikker. De enkleste metoder til at visualisere lignin og andre aromater ansætte ultraviolet (UV) lys. Excitation af aromatiske molekyler med UV-lys er en gammel teknik, men det er stadig en af ​​de hurtigste metoder til visualisering af lignin. Men UV visualisering faktisk ikke er ideel til lignin detektion, fordi UV-lys vil excitere andre aromater. Lignin består primært af tre byggesten, de monolignols (hydroxycinnamyl alkoholer: coniferyl alkohol, sinapyl alkohol, og p-coumaryl alkohol) 1-3. Phloroglucinol plet kan give et fingerpeg om omfanget af cinnamaldehydes stede i veddet, fibre og luftrør væv 4.. Phloroglucinol er en god indikator for generelle cinnamaldehydes og kan skelne mellem cinnamaldehydes og andre aromater. De sinapyl alkohol monomerer kan påvisesog differentieres ved anvendelse af Maule pletten. Toluidinblåt O er en polykromatisk farvestof og har derfor evnen til at farve forskellige elementer i cellevæggen i forskellige farver 5,6. Den primære anvendelse af toluidinblåt O er at detektere pektin og lignin 5,6. Fordelen ved anvendelse af toluidinblåt O er, at mange elementer af cellevæggen kan visualiseres i et enkelt trin. Både Calcofluor hvid og congorødt er let at arbejde med og kan anvendes til at visualisere cellulose. Calcofluor hvide pletter cellulose, callose, og andre ikke-substituerede eller svagt substituerede β-glucaner 6-9, mens congorødt pletter direkte til β-(1 → 4)-glucaner og især cellulose 10,11. Målet med denne undersøgelse er at give enkle retningslinjer for brugen af de førnævnte farvningsteknikker at opnå billeder af høj kvalitet fra A. thaliana stængler.

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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.

Stem Indlejring og Sektionsinddeling: Brugen af den hjemmelavede plast støber at integrere stænglerne i 7% agarose vist sig at være hurtig og let (figur 1). De to dele (A og B, figur 1) den integrerede hætteglas systemet gør det nemt at nemt slipper stilken indlejret i agarose som agarose ikke klæber til hætteglassets dele, som er inaktiv, holde systemet rent. Hætteglassene kan genbruges flere gange i årevis. Den bekvemmelighed for at lagre den integrerede stilken gør o...

Watch this Scientific Journal Video about Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements at JoVE.com

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.

Histokemisk farvning af<em&gt; Arabidopsis thaliana</em&gt; Sekundært Cell vægelementer

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

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Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

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

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

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::ÃƒÅ½Ã‚Â²-glucuronidase Reporter Gene Expression

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Effektiv og hurtig Isolering af Debuterende embryoner fra<em&gt; Arabidopsis thaliana</em&gt; Frø

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Metabolisk mærkning og membranfraktionering for Komparativ proteomiske analyse af<em&gt; Arabidopsis thaliana</em&gt; Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

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High Resolution Kvantificering af krystallinsk cellulose Ophobning i<em&gt; Arabidopsis</em&gt; Roots til Monitor Tissue-specifikke Cell Wall Ændringer

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Analyse af Arabidopsis thaliana vækst adfærd i forskellige lys kvaliteter

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

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Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells (Video) | JoVE 

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

An Improved Method to Measure Pollen Hydration Profiles in Arabidopsis thaliana

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

A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana (Video) | JoVE 

En højopløsning, enkeltkornet, in vivo pollenhydrering 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 ...

Growth of Arabidopsis thaliana Plants and Preparation of Flowers for Pollen Hydration Assay

Growth of Arabidopsis thaliana Plants and Preparation of Flowers for Pollen Hydration Assay  (Video) | JoVE 

Vækst af Arabidopsis thaliana planter og forberedelse af blomster til pollenhydrering assay

﻿Preparation of Dual-Colored F1 Transgenic Arabidopsis thaliana

ï»¿Preparation of Dual-Colored F1 Transgenic Arabidopsis thaliana (Video) | JoVE 

Fremstilling af dobbeltfarvet F1 transgen arabidopsis thaliana