Name: mycorrhizal maps as a tool to explore colonization patterns and fungal strategies in the roots of festuca rubra and zea mays
Uploaded: 2022-08-26T13:00:00
Description: Watch this Scientific Journal Video about Mycorrhizal Maps as a Tool to Explore Colonization Patterns and Fungal Strategies in the Roots of Festuca rubra and Zea mays at JoVE.com

Dette papir bruger data fra to ph.d.-undersøgelser inden for det tematiske område af "Corn Mycorrhizal Patterns Driven by Agronomic Inputs", udført af Victoria Pop-Moldovan, og "Mycorrhizal Status and Development of Colonization in Mountain Grassland Dominant Species", udført af Larisa Corcoz, under koordinering af prof. Dr. Roxana Vidican.

1. Udvælgelse af biologisk materiale, rodprøvetagning og opbevaring
<ol><li>Saml hele roden af planter med en skovl (figur 1A) separat for hver variant og repliker. Fjern forsigtigt de store jordaggregater fra rødderne med hånden. Vask hele rodsystemet og mål det på en skala med 1 cm x 1 cm celler (figur 1B). Skær rødderne separat for hver plante, og læg dem i en plastikpose.</li>
	<li>Saml alle de rene rødder fra hver plante i en pl...

<ol>
	<li>Trivedi, P., Leach, J. E., Tringe, S. G., Sa, T., Singh, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-microbiome+interactions:+From+community+assembly+to+plant+health.">Plant-microbiome interactions: From community assembly to plant health.</a> Nature Reviews Microbiology. 18 (11), 607-621 (2020).</li><li>Jeffries, P., Barea, J. M., Hock, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4+Arbuscular+Mycorrhiza:+A+Key+Component+of+Sustainable+Plant-Soil+Ecosystems.">4 Arbuscular Mycorrhiza: A Key Component of Sustainable Plant-Soil Ecosystems.</a> The Mycota. IX Fungal Associations. , 51-75 (2012).</li><li>Parniske, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arbuscular+mycorrhiza:+the+mother+of+plant+root+endosymbioses.">Arbuscular mycorrhiza: the mother of plant root endosymbioses.</a> Nature Reviews Microbiology. 6 (10), 763-775 (2008).</li><li>Gianinazzi, S., 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=Agroecology:+The+key+role+of+arbuscular+mycorrhizas+in+ecosystem+services.">Agroecology: The key role of arbuscular mycorrhizas in ecosystem services.</a> Mycorrhiza. 20 (8), 519-530 (2010).</li><li>Lee, E. -. H., Eo, J. -. K., Ka, K. -. H., Eom, A. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+arbuscular+mycorrhizal+fungi+and+their+roles+in+ecosystems.">Diversity of arbuscular mycorrhizal fungi and their roles in ecosystems.</a> Mycobiology. 41 (3), 121-125 (2013).</li><li>Zhang, Y., Zeng, M., Xiong, B., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological+significance+of+arbuscular+mycorrhiza+biotechnology+in+modern+agricultural+system.">Ecological significance of arbuscular mycorrhiza biotechnology in modern agricultural system.</a> Ying Yong Sheng Tai Xue Bao = The Journal of Applied Ecology. 14 (4), 613-617 (2003).</li><li>Shah, A. A., 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=Effect+of+endophytic+Bacillus+megaterium+colonization+on+structure+strengthening,+microbial+community,+chemical+composition+and+stabilization+properties+of+Hybrid+Pennisetum.">Effect of endophytic Bacillus megaterium colonization on structure strengthening, microbial community, chemical composition and stabilization properties of Hybrid Pennisetum.</a> Journal of the Science of Food and Agriculture. 100 (3), 1164-1173 (2020).</li><li>Souza, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Arbuscular Mycorrhizal Fungi. , (2015).</li><li>Sun, X. -. G., Tang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+four+routinely+used+methods+for+assessing+root+colonization+by+arbuscular+mycorrhizal+fungi.">Comparison of four routinely used methods for assessing root colonization by arbuscular mycorrhizal fungi.</a> Botany. 90 (11), 1073-1083 (2012).</li><li>Smith, S., Read, D., Smith, S. E., Read, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonization+of+Roots+and+Anatomy+of+Arbuscular+Mycorrhiza.">Colonization of Roots and Anatomy of Arbuscular Mycorrhiza.</a> Mycorrhizal Symbiosis. , 42-90 (2008).</li><li>Stoian, V., 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=Sensitive+approach+and+future+perspectives+in+microscopic+patterns+of+mycorrhizal+roots.">Sensitive approach and future perspectives in microscopic patterns of mycorrhizal roots.</a> Scientific Reports. 9 (1), 10233 (2019).</li><li>Pop-Moldovan, V., 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=Divergence+in+corn+mycorrhizal+colonization+patterns+due+to+organic+treatment.">Divergence in corn mycorrhizal colonization patterns due to organic treatment.</a> Plants. 10 (12), 2760 (2021).</li><li>Corcoz, L., 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=Mycorrhizal+patterns+in+the+roots+of+dominant+Festuca+rubra+in+a+high-natural-value+grassland.">Mycorrhizal patterns in the roots of dominant Festuca rubra in a high-natural-value grassland.</a> Plants. 11 (1), 112 (2021).</li><li>Corcoz, L., 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=Deciphering+the+colonization+strategies+in+roots+of+long-term+fertilized+Festuca+rubra.">Deciphering the colonization strategies in roots of long-term fertilized Festuca rubra.</a> Agronomy. 12 (3), 650 (2022).</li><li>Stoian, V., Florian, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycorrhiza+-+Benefits,+influence,+diagnostic+method.">Mycorrhiza - Benefits, influence, diagnostic method.</a> Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Agriculture. 66 (1), 2009 (2009).</li><li>Prates J&#250;nior, P., 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=Agroecological+coffee+management+increases+arbuscular+mycorrhizal+fungi+diversity.">Agroecological coffee management increases arbuscular mycorrhizal fungi diversity.</a> PLoS One. 14 (1), 0209093 (2019).</li><li>Rillig, M. C., 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=Why+farmers+should+manage+the+arbuscular+mycorrhizal+symbiosis.">Why farmers should manage the arbuscular mycorrhizal symbiosis.</a> New Phytologist. 222 (3), 1171-1175 (2019).</li><li>Rillig, M. C., 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=Towards+an+integrated+mycorrhizal+technology:+Harnessing+mycorrhiza+for+sustainable+intensification+in+agriculture.">Towards an integrated mycorrhizal technology: Harnessing mycorrhiza for sustainable intensification in agriculture.</a> Frontiers in Plant Science. 7, 1625 (2016).</li><li>Bhale, U. N., Bansode, S. A., Singh, S., Gehlot, P., Singh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifactorial+Role+of+Arbuscular+Mycorrhizae+in+Agroecosystem.">Multifactorial Role of Arbuscular Mycorrhizae in Agroecosystem.</a> Fungi and their Role in Sustainable Development: Current Perspectives. , 205-220 (2018).</li><li>Khaliq, A., 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=Integrated+control+of+dry+root+rot+of+chickpea+caused+by+Rhizoctonia+bataticola+under+the+natural+field+condition.">Integrated control of dry root rot of chickpea caused by Rhizoctonia bataticola under the natural field condition.</a> Biotechnology Reports. 25, 00423 (2020).</li><li>Vaida, I., P&#259;curar, F., Rotar, I., Tomo&#537;, L., Stoian, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+diversity+due+to+long-term+management+in+a+high+natural+value+grassland.">Changes in diversity due to long-term management in a high natural value grassland.</a> Plants. 10 (4), 739 (2021).</li><li>. The International Collection of (Vesicular) Arbuscular Mycorrhizal Fungi Available from: <a target="_blank" href="https://invam.wvu.edu/collection">https://invam.wvu.edu/collection</a> (2022)</li><li>. The International Bank for the Glomeromycota Available from: <a target="_blank" href="https://www.i-beg.eu/">https://www.i-beg.eu/</a> (2022)</li></ol>

Undersøgelser af mycorrhizal kolonisering er afgørende for ny strategiudvikling på det agronomiske område. Potentialet for flere dyrkede planter til at danne en symbiotisk tilknytning til arbuskulære mycorrhizas gjorde dem til en vigtig komponent i agroøkosystemets bæredygtige udvikling og opretholdelsen af dets sundhed 16,17,18,19,20. Der er således b...

Arbuskulære mycorrhiza (AM) svampe er en kategori af jordbårne endofytter, der konstant er et område af interesse for forskere. Deres tilstedeværelse i de fleste planters rødder og deres involvering i næringsstofcyklusser gør dem til vitale komponenter i stabiliteten af ethvert økosystem, hvor urteagtige planter er til stede 1,2. Gennem deres ekstra-radikulære mycelium fungerer AM som en svampeforlængelse for planterødder, især i svært tilgængelige områder3. Hovedaktiviteten er i værtsplanterødder, hvor AM udvikler store hyfernetværk og specifikke intracellulære strukturer kaldet arbuscules. Manglen på værtsspecificitet gør det muligt for symbionten at kolonisere flere arter på samme tid. Denne evne giver AM rollen som ressourceallokering og næringsstofregulering i økosystemet; svampen giver også støtte til planteoverlevelse og hjælper med plantens ydeevne 4,5,6,7. AM-arters reaktion på værtsrødder er synlig i forlængelsen og placeringen af det intraradikulære mycelium og tilstedeværelsen og formen af arbuscules udviklet intracellulært. De intracellulære arbuscules fungerer som et udvekslingspunkt mellem de to symbionter og repræsenterer områder præget af hurtige overførselsprocesser. De strukturer, som AM producerer, er artsafhængige, og ud over arbuscules i rødderne udvikler de også vesikler, sporer og hjælpeceller.
Der er mange udfordringer i vurderingen af AM-symbionter i planterødder 8,9. Den første er deres konstante udvikling i hele vegetationsperioden for værter, hvilket fører til flere ændringer i hyphal arbuskulær struktur. De forskellige stadier af arbuskulær vækst, op til deres sammenbrud, er tydeligt til stede i rødder, men de senescent AM-strukturer fordøjes undertiden, hvilket gør dem kun delvist synlige10. Den anden udfordring er repræsenteret af farvningsmetoden og protokollen, den store mangfoldighed af rodsystemer, dimensionen af deres celler og forskellene i tykkelse, hvilket gør det svært at foreslå en samlet metode. Den sidste udfordring er repræsenteret ved vurdering og scoring af AM-kolonisering. Der er mange metoder, der scorer AM med forskellige grader af objektivitet, og de fleste af dem er stadig begrænset til mikroskopiteknikker. De enkle er baseret på tilstedeværelsen / fraværet af strukturer i rodbarken, mens de mere komplekse er baseret på visuel scoring og brugen af koloniseringsklasser med integration af hyppigheden og intensiteten af koloniseringsfænomenet. Der er produceret mange data i de sidste årtier om mycorrhizal status for flere arter, men de fleste af metoderne er begrænset til den observerede værdi af kolonisering uden at pege på den reelle position af hver struktur i rodbarken. Som et svar på nødvendigheden af mere nøjagtige resultater om AM-kolonisering blev der udviklet en metode baseret på mikroskopisk analyse af mycorrhizale mønstre (MycoPatt) i rødder til at samle de detaljerede mycorrhizalkort i digital form11. Metoden tillader også den objektive beregning af koloniseringsparametre og bestemmelsen af den faktiske position af hver struktur i roden.
Placeringen af AM-svampestrukturerne kan være vigtig for at besvare følgende to spørgsmål. Den første er relateret til analysen af koloniseringen i et bestemt øjeblik fra vegetationscyklussen af en plante. I denne sammenhæng er det meget nyttigt at observere den arbuskulære / vesikel overflod, rapportere, hvordan de er placeret i roden, og give et meget klart koloniseringsbillede og parametre. Den anden er relateret til påvisning af svampestrategi og dens orientering og endda prognosen for dens fremtidige udvikling. En anvendelse af MycoPatt kan være til planter, der analyseres dagligt, hver 2-3 dage, ugentligt eller under forskellige vækststadier. I denne sammenhæng er placeringen af vesikler / arbuscules vigtig for bedre at forstå den biologiske mekanisme for AM-kolonisering. Disse parametre og observationer er meget nyttige til at supplere de matematiske parametre.
Formålet med denne artikel er at demonstrere MycoPatt-systemets evne til at udforske det oprindelige AM-svampekoloniseringspotentiale og strategi i Zea mays (majs) rødder i forskellige udviklingsstadier og i Festuca rubra (rød svingel) rødder under forskellige langsigtede befrugtningsbetingelser. For at opfylde målet blev to store databaser fra to eksperimenter analyseret. Majseksperimentet blev etableret ved Cojocna (46°44′56" lat. N og 23°50′0" lang. E), i den eksperimentelle didaktiske gård ved University of Agricultural Sciences and Veterinary Medicine Cluj på et phaeoziom med en leragtig teksturjord12. Rødsvingeleksperimentet er en del af et større forsøgssted, der blev etableret i 2001 i Ghețari, Apuseni-bjergene (46°49'064" lat. N og 22°81'418'' lang. E), på en preluvosol (terra rossa) jordtype13,14. Majs blev samlet i fem forskellige vækstfænofaser12: B1 = 2-4 blade (som kontrolpunkt for starten af mycorrhizal kolonisering); B2 = 6 blade; B3 = 8-10 blade; B4 = kolbdannelse; B5 = fysiologisk modenhed. Fra 2-4 blade trin (A0) blev der anvendt en organisk behandling, hvilket resulterede i en to-gradueringsfaktor (A1 = kontrol og A2 = behandlet). Rødder af rødsvingel blev indsamlet ved blomstring fra et forsøg med fem langtidsbefrugtninger13,14: V1 = kontrol, ikke-befrugtet; V2 = 10 t·ha-1 gødning; V3 = 10 t·ha-1 husdyrgødning + N 50 kg·ha-1, P 2 O5 25 kg·ha-1, K2O 25 kg·ha-1; V4 = N 100 kg·ha-1, P 2 O5 50 kg·ha-1, K2O 50 kg·ha-1; V5 = 10 t·ha-1 husdyrgødning + N 100 kg·ha-1, P 2 O5 50 kg·ha-1, K2O 50 kg·ha-1. Fem planter blev indsamlet i hvert udviklingsstadium fra hver befrugtningsvariant. Farvningsprotokollerne og deres ydeevne med hensyn til prøvebehandlingstid og farvningskvalitet blev analyseret. Forholdet mellem AM-hyferudvikling og tilstedeværelsen af dets strukturer i rødder blev analyseret separat for hver art og fortsatte med identifikationen af de mest tilladelige rødder til kolonisering. De specifikke koloniseringsmønstre for hvert rodsystem blev analyseret baseret på koloniseringskort og værdien af AM-parametre.
Majs er en årlig plante, hvilket indebærer kontinuerlig vækst af rødderne, og det var hovedårsagen til at anvende MycoPatt i vækststadierne. Rødsvingel er en flerårig plante fra et græsareal behandlet i lang tid med forskellige gødninger. Dens rødder har en kortere udvikling på 1 år, og anthesis betragtes som vegetationspunktet, når planten ændrer sit stofskifte fra vegetativ til generativ. For at fange disse planter i disse intense aktivitetsperioder blev ovennævnte tidspunkter valgt. Prøveudtagning i vegetationsperioden er vanskelig for denne art, når den dyrkes i naturlige græsarealer.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Apple vinegar 5%</td>
			<td>FABRICA DE CONSERVE RAURENI S.R.L.</td>
			<td>O&#538;ET DE MERE</td>
			<td>https://www.raureni.ro/ro-ro/produs/otet-de-mere</td>
		</tr>
		<tr>
			<td>Blue Ink</td>
			<td>Pelikan</td>
			<td>4001</td>
			<td>https://www.pelikan.com/pulse/Pulsar/ro_RO.Store.displayStore.224848./cerneal%C4%83-4001-de-la-pelikan</td>
		</tr>
		<tr>
			<td>Cover slips</td>
			<td>Menzel-Glaser</td>
			<td>D 263 M</td>
			<td>https://si.vwr.com/store/product/20545757/cover-glasses-menzel-glaser</td>
		</tr>
		<tr>
			<td>Forceps, PMP</td>
			<td>Vitalab</td>
			<td>9.171 411</td>
			<td>http://shop.llg.de/info881_Forceps_PMP_lang_UK. 
			htm?UID=55005bf838d8000000000000 
			&#38;amp;OFS=33</td>
		</tr>
		<tr>
			<td>Glass jar 47 mL</td>
			<td>Indigo Cards</td>
			<td>BORCAN 47 ML HEXAGONAL</td>
			<td>https://indigo.com.ro/borcan-47-ml-hexagonal</td>
		</tr>
		<tr>
			<td>Laminating Pouches</td>
			<td>Peach</td>
			<td>PP525-08</td>
			<td>Business Card (60x90mm) / https://supremoffice.ro/folie-laminare-60x90mm-125mic-carte-vizita-100-top-peach-pp525-08-510328</td>
		</tr>
		<tr>
			<td>Microflow Class II ABS Cabinet</td>
			<td>Bioquell UK Ltd</td>
			<td>Microflow Class II ABS Cabinet</td>
			<td>http://www.laboratoryanalysis.co.uk/graphics/products/034_11%20CLASS%202BSC%20(STD).pdf</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Deltalab</td>
			<td>D100001</td>
			<td>https://distrimed.ro/lame-microscop-matuite-la-un-capat-26x76-mm-deltalab/?utm_source=Google%20Shopping&#38;utm_campaign= 
			google%20shopping%20distrimed&#38;utm_medium=cpc&#38; 
			utm_term=1647&#38;gclid=CjwKCAjwu 
			YWSBhByEiwAKd_n_odzr8CaCXQ 
			hl9VQkAB3p-ODo2Ssuou9cnoRtz1Gb 
			xsjqPY7F05HmhoCj6oQAvD_BwE</td>
		</tr>
		<tr>
			<td>Microsoft Office 365</td>
			<td>Microsoft</td>
			<td>Office 365</td>
			<td>Excel and Powerpoint; spreadsheet and presentation</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Oltchim</td>
			<td>01-2119457892-27-0065</td>
			<td>http://www.sodacaustica.com.ro/pdf/fisa-tehnica-soda-caustica.pdf</td>
		</tr>
		<tr>
			<td>Nitrile gloves</td>
			<td>SemperGuard</td>
			<td>816780637</td>
			<td>https://www.sigmaaldrich.com/RO/en/product/aldrich/816780637?gclid=CjwKCAjwuYWSBhByEiwAKd 
			_n_rmo4RRt8zBql7ul8ox 
			AAYhwhxuXHWZcw4hlR 
			x0Iro_4IyVt69aFHRoCmd 
			wQAvD_BwE</td>
		</tr>
		<tr>
			<td>Optika camera</td>
			<td>OPTIKA</td>
			<td>CP-8; P8 Pro Camera, 8.3 MP CMOS, USB 3.0</td>
			<td>https://www.optikamicroscopes.com/optikamicroscopes/product/c-p-series/</td>
		</tr>
		<tr>
			<td>Optika Microscope</td>
			<td>OPTIKA</td>
			<td>B383pL</td>
			<td>https://www.optikamicroscopes.com/optikamicroscopes/product/b-380-series/</td>
		</tr>
		<tr>
			<td>Protective mask FFP3</td>
			<td>Hermes Gift</td>
			<td>HERMES000100</td>
			<td>EN 149-2001+A1:2009 / https://www.emag.ro/set-10-masti-de-protectie-respiratorie-hermes-gift-ffp3-5-straturi-albe-hermes000100/pd/DTZ8CXMBM/#specification-section</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Cutfix</td>
			<td>9409814</td>
			<td>https://shop.thgeyer-lab.com/erp/catalog/search/search.action;jsessionid=C258CA 
			663588CD1CBE65BF 
			100F85241B?model.query=9409809</td>
		</tr>
		<tr>
			<td>White wine vinegar 9%</td>
			<td>FABRICA DE CONSERVE RAURENI S.R.L.</td>
			<td>O&#538;ET DE VIN ALB</td>
			<td>https://www.raureni.ro/ro-ro/produs/otet-de-vin-alb</td>
		</tr>
	</tbody>
</table>

mycorrhizal maps as a tool to explore colonization patterns and fungal strategies in the roots of festuca rubra and zea mays

Arbuskulære mycorrhizal svampe er symbionter i planternes rødder. Deres rolle er at opretholde værtsudvikling og opretholde den ernæringsmæssige ligevægt i økosystemerne. Koloniseringsprocessen er afhængig af flere faktorer som jordøkologi, svampens og værtens genetiske mangfoldighed og agronomisk praksis. Deres synkroniserede virkning fører til udviklingen af et komplekst hyphalnetværk og fører til sekundær udvikling af vesikler og arbuscules i rodcellerne. Formålet med denne forskning var at analysere effektiviteten af mycorrhizal mønstre (MycoPatt) metode til positionering af svampestrukturer i rødderne af Festuca rubra og Zea mays. Et andet mål var at udforske svampekoloniseringsstrategien som afsløret af mycorrhizalkort over hver art. Erhvervelsen og samlingen af flere mikroskopiske billeder tillader mycorrhizal koloniseringsvurdering i både majs- og rødsvingelplanter for at give information om den realistiske position af de udviklede strukturer. De observerede mykorrhizale mønstre fremhæver den variable effektivitet af hver plante med hensyn til at udvikle forbindelser med jordsymbiotiske svampe forårsaget af anvendte behandlinger og vækststadium. Mycorrhizal detaljerede kort opnået gennem MycoPatt-metoden er nyttige til tidlig påvisning af planteeffektivitet i symbiotisk erhvervelse fra jorden.

Den korrekte brug af røddernes blide knusningsmetode efter farvningsprocedurerne giver gode detaljer om mycorrhizale strukturer, både for Zea mays (figur 8A-C) og Festuca rubra (figur 9A-E), god kontrast mellem mycorrhizal strukturer og rodceller og en bekræftelse af stelen på grund af den blå farve. Hvis rydnings- og farvningsprocedurerne ikke lykkes, er rodprøver svære...

Watch this Scientific Journal Video about Mycorrhizal Maps as a Tool to Explore Colonization Patterns and Fungal Strategies in the Roots of Festuca rubra and Zea mays at JoVE.com

Mycorrhizal Maps as a Tool to Explore Colonization Patterns and Fungal Strategies in the Roots of Festuca rubra and Zea mays

Protokollen her beskriver metoderne til vurdering af de arbuskulære mycorrhizal koloniseringsmønstre og strategi i rødder for to arter: Zea mays og Festuca rubra. Brugen af MycoPatt-metoden tillader beregning af parametre, omdannelse af mykorrhizastrukturer til digitale data og kortlægning af deres reelle position i rødder.

Mycorrhizal kort som et værktøj til at udforske koloniseringsmønstre og svampestrategier i rødderne af Festuca rubra og Zea mays

Arbuscular mycorrhizal fungi are symbionts in the roots of plants. Their role is to sustain host development and maintain the nutritional equilibrium in ...

MycoPatt metode tillader en dybdegående udforskning af arbuskulær mycorrhizal kolonisering i rødder. Mycorrhizal kort peger på svampeudvidelse og præsenterer den reelle position af hver struktur som et specifikt mønster. Mycorrhizal kolonisering analyseres objektivt ved 1% opløsning.Hvert rodsegment konverteres automatisk til en digital matrix og genererer en omfattende database med koloniseringsparametre. Til at begynde med skal du fjerne rødderne fra køleskabet og placere dem på en arbejdsplatform til optøning ved stuetemperatur. For at udføre rodrensning skal du placere alle rødder fra en plante i en 30 til 50 ml krukke, derefter forberede en 10% natriumhydroxidopløsning med ledningsvand og hæld den i krukken, indtil den helt dækker rødderne.Ryst krukken kraftigt i et til to minutter for homogen spredning af rydningsopløsning i rødderne. Lad rødderne forblive i rydningsopløsningen i 48 timer. Til rodskylning skal du føre indholdet af krukken gennem en sigte.Skyl rødderne flere gange i ledningsvand, indtil rydningsopløsningen er helt fjernet. Placer derefter de skyllede rødder i en ren krukke til farvning. Forbered derefter en fem til fem procent blækeddikeopløsning med ledningsvand.Hæld det i krukken, indtil det dækker rødderne og ryst det i et til to minutter. Efter 48 timer til delvis affarvning skylles de farvede rødder i ledningsvand i et til to minutter. Ryst krukken kraftigt for at fjerne den ekstra farvningsopløsning.Gentag proceduren, indtil rødderne er klare til mikroskopisk evaluering. Til rodsegmentering skal du placere de farvede rødder på et skaleret skærebræt. Skær rødderne i segmenter på en centimeter.Vælg 15 segmenter for hver variant. Spred derefter rødderne på et dias til segmentforberedelse, brug en lamineringspose til at dække rødderne og knus dem forsigtigt, startende fra en kant. Brug et blødt plastværktøj til at vise rødderne på diaset.Fjern forsigtigt lamineringsposen, og dæk prøven med en dæksel. Tilsæt vand til et hjørne af rutsjebanen med en pipette, og lad vandet langsomt sprede sig på rutsjebanen. Fjern det ekstra vand med et papirhåndklæde.Til mikroskopisk analyse skal du bruge et mikroskop udstyret med et kamera med god opløsning. Analyser diasene startende fra en ekstremitet. Fang hvert mikroskopisk felt.Brug 10 eller 40x forstørrelse til tykke rødder og 40x til tynde rødder. Efter optagelse skal du omdøbe hvert billede med en kode, der tillader den rigtige eftermontering af roddele. Brug præsentationssoftware til at designe et tegnebræt til billedsamling, indstil bredden to til tre centimeter bredere end billedbredden.Når du har taget 15 billeder, skal du tilføje alle optagne billeder fra et segment i rækkefølgen af deres optagelse, startende med et til 15, og rekonstruere hele længden af rodsegmentet. Brug derefter den lodrette justering til at justere billederne i midten og sikre, at hvert billede følger det forrige. Placer et gitter på 10 x 150 celler på alle billeder for at dække hele rodsegmentet.Derudover skal du placere et 10 x 10 gitter på hvert billede og i hver celle i dette gitter, indsætte et tal fra et til seks, hvis en arbuskulær mycorrhizal eller AM-struktur er synlig, eller lade være tom, hvis der ikke er nogen AM-struktur til stede. Tilføj en tabel for et gitter med 10 celler i bredden og 150 celler i længden. Skift tabellen med dimensioner til billedernes bredde, og skift derefter tabellens længde, så den omfatter alle billederne.For at score mycorrhizal kolonisering skal du bruge det unikke nummer til at score hver type struktur som beskrevet i mycorrhizal mønstermetoden. Brug en til hyfer, to til arbuscules, tre til vesikler, fire til sporer, fem til hjælpeceller og seks til indgangspunkter. Scor hver observeret mycorrhizal struktur fra hver celle i de tidligere anvendte gitter.Efter scoring skal du indsætte alle de opnåede scoringer i MycoPatt-regnearket. Brug funktionen kopiér indsæt til at overføre alle scores fra præsentationen til det første ark med navnet Rådata. Til den primære analyse skal du bruge det tredje ark med navnet Parametre til at visualisere resultaterne som procenter separat i tre former.Brug kolonnerne A til K til at analysere det vandrette billede af kolonisering, kolonnerne M til W til det lodrette billede af koloniseringen og kolonnerne Y til AI til at analysere den tværgående kolonisering for hver af de 15 10 x 10 kvadrater og den endelige gennemsnitlige kolonisering. Anvend derefter definitionerne og formlerne, der er specifikke for hver parameter. Til produktion og udvinding af mycorrhizal kort skal du visualisere billedet opnået ved omdannelse af mycorrhizal strukturkode til farver i det andet ark med navnet Graphs.Eksporter derefter det resulterende billede i grafarket som et billede, og brug farvekoden i forklaringen til at analysere mycorrhizale mønstre. Til mycorrhizal kortanalyse skal du identificere de vigtigste strukturer og deres samling. Beskriv derefter det mycorrhizale koloniseringsmønster, der er observeret i de analyserede rødder, efterfulgt af en koloniseringsstrategi baseret på observeret strukturel udvikling i roden, forgreningsmønsteret og arbuskulær vesikeludvikling.Denne protokol giver detaljerne om mycorrhizal strukturer med god kontrast mellem mycorrhizal strukturer og rodceller til Zea mays og Festal rubra. De mislykkede rydnings- og farvningsprocedurer resulterede i uklare mikroskopiske billeder af mykorrhizale strukturer i Festal rubra og Zea mays. Mycorrhizal koloniseringsmønstre tillod en fuldstændig udforskning af koloniseringsmekanismen, denne metode giver en dyb, lille skala udforskning af koloniseringsmønstre og strategier for Zea mays og Festuca rubra med et yderligere visuelt udtryk for koloniseringsparametre.I Zea mays blev der observeret stærkt svingende koloniseringspotentiale afhængigt af plantens udviklingsstadium, men i Festuca rubra forekommer koloniseringsprocesserne inde i rødderne, og udviklingen af hyphalnetværk var korreleret med en lav udviklingshastighed af rødderne. MycoPatt er velegnet til kortlægning af den arbuskulære mycorrhizal kolonisering i enhver plante. Det er også nyttigt at udforske koloniseringsmekanismen fuldt ud og vurdere svampestrategi langs rødderne.

mycorrhizal-maps-as-tool-to-explore-colonization-patterns-fungal

Research

JoVE Journal

Biology

Mycorrhizal Maps as a Tool to Explore Colonization Patterns and Fungal Strategies in the Roots of Festuca rubra and Zea mays

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.
On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision 2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) 4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy 6. This powerful technique has been implemented to study many aspects of cell biology 5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. 
Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from &le;5 s up to several hours, depending on the nature of the microinjected sample.

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Brug SecM Arrest Sequence som et redskab til at isolere ribo bundne polypeptider

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

En guide til Modern Quantitative Fluorescent Western Blotting med fejlfinding Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

C-FOS Protein Immunohistologisk Detection: Et nyttigt værktøj som en markør for Central involverede veje i specifikke fysiologiske reaktioner<em&gt; In vivo</em&gt; og<em&gt; Ex vivo</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

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

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 containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

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

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

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 ...

Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves

Growth analyses are often used in plant science to investigate contrasting genotypes and the effect of environmental conditions. The cellular aspect of these analyses is of crucial importance, because growth is driven by cell division and cell elongation. Kinematic analysis represents a methodology to quantify these two processes. Moreover, this technique is easy to use in non-specialized laboratories. Here, we present a protocol for performing a kinematic analysis in monocotyledonous maize (Zea mays) leaves. Two aspects are presented: (1) the quantification of cell division and expansion parameters, and (2) the determination of the location of the developmental zones. This could serve as a basis for sampling design and/or could be useful for data interpretation of biochemical and molecular measurements with high spatial resolution in the leaf growth zone. The growth zone of maize leaves is harvested during steady-state growth. Individual leaves are used for meristem length determination using a DAPI stain and cell-length profiles using DIC microscopy. The protocol is suited for emerged monocotyledonous leaves harvested during steady-state growth, with growth zones spanning at least several centimeters. To improve the understanding of plant growth regulation, data on growth and molecular studies must be combined. Therefore, an important advantage of kinematic analysis is the possibility to correlate changes at the molecular level to well-defined stages of cellular development. Furthermore, it allows for a more focused sampling of specified developmental stages, which is useful in case of limited budget or time.

Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves

Quantifying cell division and expansion is of crucial importance to the understanding of whole-plant growth. Here, we present a protocol to calculate cellular parameters determining maize leaf growth rates and highlight the use of these data for investigating molecular growth regulatory mechanisms by directing developmental stage-specific sampling strategies.

Kinematisk Analyse af celledeling og Expansion: Kvantificering af Cellular Grundlag for vækst og Sampling Developmental zoner i<em&gt; Zea mays</em&gt; Blade

Growth analyses are often used in plant science to investigate contrasting genotypes and the effect of environmental conditions. The cellular aspect of ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

Light Sheet Fluorescens mikroskopi af planterødder Dyrkning på overfladen af ​​en gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Magnetisk aktiveret celle sortering strategier for at isolere og rense Synovial væske-afledte mesenkymale stamceller fra en kanin Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological samples, designed for high-resolution magnetic resonance imaging (MRI), also referred to as MR microscopy (MRM). It may be used at pre-clinical MRI spectrometers, demonstrated on Medicago truncatula root samples. Microcoils increase sensitivity by matching the size of the RF resonator to the size of the sample of interest, thereby enabling higher image resolutions in a given data acquisition time. Due to the relatively simple design, solenoidal microcoils are straightforward and cheap to construct and can be easily adapted to the sample requirements. Systematically, we explain how to calibrate new or home-built microcoils, using a reference solution. The calibration steps include: pulse power determination using a nutation curve; estimation of RF-field homogeneity; and calculating a volume-normalized signal-to-noise ratio (SNR) using standard pulse sequences. Important steps in sample preparation for small biological samples are discussed, as well as possible mitigating factors such as magnetic susceptibility differences. The applications of an optimized solenoid coil are demonstrated by high-resolution (13 x 13 x 13 &#956;m3, 2.2 pL) 3D imaging of a root sample.

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

A protocol to study biological tissue at high spatial resolution using ultra-high field magnetic resonance microscopy (MRM) using microcoils is presented. Step-by-step instructions are provided for characterizing the microcoils. Finally, optimization of imaging is demonstrated on plant roots.