Wir sind dankbar, Herr Akira Murakami der Abteilung für Angewandte Biowissenschaften, Toyo University und Herr Koudai Taniguchi Gunma Agricultural Technology Center für ihre technische Unterstützung. Wir sind auch Professor Shosaku Kashiwada und Dr. Uma Maheswari Rajagopalan, Toyo University für ihre Anregungen dankbar. Diese Studie wurde teilweise durch das Forschungszentrum für Leben und Umweltwissenschaften, Toyo University unterstützt.

Hinweis: Ipecac (C. Ipecacuanha) wurde in dieser Studie verwendet, weil es die Analyse der endogenen Phytohormone erleichtert.
(1) Wachstumsbedingungen induzieren zufällige Triebe von Ipecac
<ol><li>Bereiten Sie Phytohormon-freie B5 Medium bis pH 5,710eingestellt zu, und 0,2 % Gellan Gum. Sterilisieren Sie durch Autoklavieren.</li>
	<li>Gießen Sie 25 mL des Mediums autoklaviert in eine sterile Petrischale (90 mm × 20 mm).</li>
	<li>8 mm Internodien Segmente von Ipecac Pflänzchen mit einem chirurgischen Skalpell mit Klinge Nr. 22 auf eine sterile Acrylplatte, und auf dem Medium (Abbildung 1).</li>
	<li>Kultur auf Phytohormon-freie B5 Medium bei 24 ° C unter 15 µmol m−2 s−1 des Lichts in einem 14-h Licht/10-h dunklen Zyklus für 5 Wochen.</li>
	<li>Regionen zu identifizieren ich (apikalen) bis IV (basal) in jedem Segment (Abbildung 1B). Die Anzahl der zufällige Triebe > 0,3 mm Länge in den einzelnen Regionen unter dem Mikroskop einmal pro Woche.</li>
</ol>(2) Extraktion und Reinigung von Phytohormonen
<ol><li>Setzen Sie eine 5 mm Zirkonia Perle in jeder der vier 2-mL-Probenröhrchen.</li>
	<li>Schneiden Sie die Segmente in Regionen I bis IV (je 2 mm in der Länge) mit einem chirurgischen Skalpell auf eine sterile Acrylplatte.</li>
	<li>Sammeln Sie acht Segmente der einzelnen Regionen in separaten Probenröhrchen (10-30 mg Frischgewicht).</li>
	<li>Wiegen, und dann Einfrieren der Proben in flüssigem Stickstoff.</li>
	<li>Die gefrorenen Proben unter Verwendung einer Perle-basierte Homogenisator zu vernichten.</li>
	<li>Aussetzen der zerkleinerten Proben in 1 mL Acetonitril mit 500 Pg der einzelnen internen Standards von Auxin und CKs (d5-IAA, d5- tZ, d5- tZR, d6- iP, d6- iPR) mit einem Vortex-Mixer.</li>
	<li>Halten Sie bei 4 ° C für 1 h und Zentrifugieren bei 3.500 × g für 5 min bei Raumtemperatur.</li>
	<li>Waschen Sie das Pellet in 80 % (V/V) Acetonitril mit Essigsäure 1 % (V/V). Zentrifuge wieder bei 3.500 × g für 5 min bei Raumtemperatur. Kombinieren Sie die Überstände (aus Schritt 2.7 und 2.8) in einer Einweg-Glasröhre.</li>
	<li>Fügen Sie 600 µL Wasser mit 1 % (V/V) Essigsäure zu jeder kombinierten überstand und verdunsten das mit einer Vakuum-Konzentrator Acetonitril.</li>
	<li>Equilibrate hydrophilen lipophil-ausgewogen (HLB) Spalte Patronen durch die Anwendung von 1 mL Acetonitril, Methanol und Wasser mit 1 % (V/V) Essigsäure.</li>
	<li>Gelten Sie eine Beispiellösung pro äquilibriert HLB Patrone.</li>
	<li>Waschen Sie die Patronen mit 1 mL Wasser mit 1 % (V/V) Essigsäure.</li>
	<li>Eluieren Sie alle Hormone mit 2 mL 80 % (V/V) Acetonitril mit 1 % (V/V) Essigsäure in einer Glasröhre.</li>
	<li>Verdunsten Sie Acetonitril in das Eluat Extrakt in Wasser mit 1 % (V/V) Essigsäure mit einem Vakuum Konzentrator zu erhalten. Hinweis: Nicht trocken Sie dies vollständig.</li>
	<li>Equilibrate gemischt-Modus, starken Kationenaustausch (MCX) Spalte Patronen durch die Anwendung 1 mL Acetonitril, 1 mL Methanol, 0,5 mL 0,1 M HCl und 1 mL Wasser mit 1 % (V/V) Essigsäure.</li>
	<li>Gelten Sie eine Beispiellösung pro äquilibriert MCX Patrone.</li>
	<li>Waschen Sie die Patronen mit 1 mL Wasser mit 1 % (V/V) Essigsäure.</li>
	<li>Eluieren Sie IAA mit 2 mL 30 % (V/V) Acetonitril mit 1 % (V/V) Essigsäure in einer Glasröhre.</li>
	<li>Waschen Sie die Patronen mit 2 mL 80 % (V/V) Acetonitril mit Essigsäure 1 % (V/V).</li>
	<li>Waschen Sie die Patronen mit 2 mL Wasser und 1 mL Wasser, 5 % wässriger Ammoniak enthält.</li>
	<li>Die CKs mit 2 mL 60 % (V/V) Acetonitril mit 5 % wässriger Ammoniak in einer Glasröhre eluieren.</li>
	<li>Verdunstet das Lösungsmittel der einzelnen Hormon-Fraktionen mit einem Vakuum Konzentrator und Lagern bei-30 ° C bis die LC-MS/MS-Analyse.</li>
</ol>(3) LC-MS/MS-Analyse der IAA und CKs
<ol><li>Jedes Hormon-Extrakt in einem Rohr mit 600 µL Methanol auflösen und die Lösung zu einem Schraube Hals völligen Heilung Fläschchen zu übertragen. Verdunsten Sie das mit einer Vakuum-Konzentrator Lösungsmittel.</li>
	<li>Auflösen der IAA Bruch in 20 µL 30 % (V/V) Acetonitril und die CK-Brüche in 20 µL Wasser mit 1 % (V/V) Essigsäure in Schraube Hals völligen Heilung Fläschchen.</li>
	<li>Analysieren Sie die Proben im positiven Ionen-Modus auf eine Triple-Quadrupol-MS-System mit einer HPLC-Anlage ausgestattet. Hinweis: Wir legen die HPLC-Bedingungen, wie in Tabelle 1aufgeführt.<ol>
<li>Für die IAA Elution verwendet einen binären Gradienten von 5-50 % Lösungsmittel B mehr als 7 min, dann erhöhen durch 98 % Lösungsmittel B 1 min warten und dann für 2 min bei 5 % Lösungsmittel B durch Nest Injektion equilibrate.</li>
		<li>Für die CK-Elution verwendet eine binäre Gefälle von 2 % - 40 % Lösungsmittel B mehr als 5 min, 40-70 % Lösungsmittel B in 7 min, dann erhöhen, indem Sie 95 % Lösungsmittel B 1 min warten und dann für 2 min bei 2 % Lösungsmittel B durch equilibrate Injektion zu verschachteln.</li>
		<li>Festlegen die Elektrospray-Ionisation (ESI)-MS-Parameter der Ionenquelle wie in Tabelle 2aufgeführt.</li>
	</ol>
</li>
	<li>Verwenden Sie die mehrere Reaktion monitoring (MRM) Übergang zur Quantifizierung der einzelnen Analyten in Tabelle 3aufgeführten.</li>
	<li>Endogene IAA und CK-Ebene gegen eine Standardkurve des Verhältnisses der unbeschrifteten Deuterium-Label Standards zu quantifizieren.</li>
</ol>

<ol>
	<li>Haberlandt, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kulturversuche+mit+isolierten+Pflanzenzellen.">Kulturversuche mit isolierten Pflanzenzellen.</a> Sitzungsber. Math.-Naturwiss. Kl. Akad. Wiss. Wien. 111, 69-92 (1902).</li><li>Skoog, F., Miller, C. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+regulation+of+growth+and+organ+formation+in+plant+tissues+cultured+in+vitro.">Chemical regulation of growth and organ formation in plant tissues cultured in vitro.</a> Symp Soc Exp Biol. 11, 118-130 (1957).</li><li>Ganeshan, S., Caswell, K. L., Kartha, K. K., Chibbar, R. N., Khachatourians, G. G., McHughen, A., Scorza, R., Nip, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Transgenic plants and crops. , 69-84 (2002).</li><li>Teshima, D., Ikeda, K., Satake, M., Aoyama, T., Shimomura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+emetic+alkaloid+by+in+vitro+culture+of+Cephaelis+ipecacuanha.">Production of emetic alkaloid by in vitro culture of Cephaelis ipecacuanha.</a> Plant Cell Rep. 7 (4), 278-280 (1988).</li><li>Chatterjee, S. K., Nandi, R. P., Ghosh, N. C., Atal, C. K., Kapur, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+and+utilixzation+of+medicinal+plants.">Cultivation and utilixzation of medicinal plants.</a> Regional Research Laboratory, Council of Scientific and Industrial Research. , 295-301 (1982).</li><li>Yoshimatsu, K., Shimomura, K., Bajaj, Y. P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biotechnology in Agriculture and Forestry 21, Medicinal and Aromatic Plants IV. , 87-103 (1993).</li><li>Ideda, K., Teshima, D., Aoyama, T., Satake, M., Shimomura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+propagation+of+Cephaelis+ipecacuanha.">Clonal propagation of Cephaelis ipecacuanha.</a> Plant Cell Rep. 7 (4), 288-291 (1988).</li><li>Yoshimatsu, K., Shimomura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+shoot+formation+on+internodal+segments+and+alkaloid+formation+in+the+regenerates+of+Cephaelis+ipecacuanha+A.+Richard.">Efficient shoot formation on internodal segments and alkaloid formation in the regenerates of Cephaelis ipecacuanha A. Richard.</a> Plant Cell Rep. 9 (10), 567-570 (1991).</li><li>Koike, I., Taniguchi, K., Shimomura, K., Umehara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+endogenous+indole-3-acetic+acid+and+cytokinins+during+adventitious+shoot+formation+in+ipecac.">Dynamics of endogenous indole-3-acetic acid and cytokinins during adventitious shoot formation in ipecac.</a> J. Plant Growth Regul. , (2017).</li><li>Gamborg, O. L., Miller, R. A., Ojima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutrient+requirements+of+suspension+cultures+of+soybean+root+cells.">Nutrient requirements of suspension cultures of soybean root cells.</a> Exp. Cell Res. 50 (1), 151-158 (1968).</li><li>Watad, 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=Adventitious+shoot+formation+from+carnation+stem+segments:+a+comparison+of+different+culture+procedures.">Adventitious shoot formation from carnation stem segments: a comparison of different culture procedures.</a> Scientia Hortic. 65 (4), 313-320 (1996).</li><li>Ajithkumar, D., Seeni, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+clonal+multiplication+through+in+vitro+axillary+shoot+proliferation+of+Aegle+marmelos+(L.)+Corr.,+a+medicinal+tree.">Rapid clonal multiplication through in vitro axillary shoot proliferation of Aegle marmelos (L.) Corr., a medicinal tree.</a> Plant Cell Rep. 17 (5), 422-426 (1998).</li><li>Tiwari, V., Tiwari, K. N., Singh, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+studies+of+cytokinins+on+in+vitro+propagation+of+Bacopa+monniera.">Comparative studies of cytokinins on in vitro propagation of Bacopa monniera.</a> Plant Cell Tiss. Org. Cult. 66 (1), 9-16 (2001).</li><li>Rao, M. S., Purohit, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+shoot+bud+differentiation+and+plantlet+regeneration+in+Celastrus+paniculatus+Willd.">In vitro shoot bud differentiation and plantlet regeneration in Celastrus paniculatus Willd.</a> Biol. Plant. 50 (4), 501-506 (2006).</li><li>Sanikhani, M., Frello, S., Serek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TDZ+induces+shoot+regeneration+in+various+Kalancho&#235;+blossfeldiana+Poelln+cultivars+in+the+absence+of+auxin.">TDZ induces shoot regeneration in various Kalancho&#235; blossfeldiana Poelln cultivars in the absence of auxin.</a> Plant Cell Tiss. Org. Cult. 85 (1), 75-82 (2006).</li><li>Yoshimoto, K., 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=Autophagy+negatively+regulates+cell+death+by+controlling+NPR1-dependent+salicylic+acid+signaling+during+senescence+and+the+innate+immune+response+in+Arabidopsis.">Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis.</a> Plant Cell. 21 (9), 2914-2927 (2009).</li><li>Schaller, G. E., Bishopp, A., Kieber, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yin-yang+of+hormones:+cytokinin+and+auxin+interactions+in+plant+development.">The yin-yang of hormones: cytokinin and auxin interactions in plant development.</a> Plant Cell. 27 (1), 44-63 (2015).</li></ol>

Die Autoren erklären, dass sie keine Interessenkonflikte.

Ermittlung die Verteilung von Phytohormonen unbedingt beteiligt Organogenese verwenden Pflanzenmaterial in denen Organogenese auf Phytohormon-freies Medium beobachtet werden kann, weil wenn Phytohormone Explantaten zur Induktion exogen zugewiesen werden Triebe oder Wurzeln, beeinflussen sie das ganze Explant, macht es schwierig, die inhärente Plastizität der Pflanzen in Zell-Differenzierung und Organogenese zu bewerten. Zufällige Triebe können auf Phytohormon-freie Kulturmedien in andere Pflanzenarten wie Dianthu...

Gottlieb Haberlandt (1854-1945) vorgeschlagen, das Konzept der "Totipotenz", von welchem Werk Zellen teilen können, unterscheiden, und ganze Pflanzen auch nach deren vorherige Differenzierung in bestimmten Zelltypen reifen Pflanzen1zu regenerieren. In der Gewebekultur wird ob Pflanze Regeneration induziert werden kann durch die Kombination und Konzentration von EXOGEN angewandte Phytohormonen in das Wachstumsmedium bestimmt. Skoog und Miller gefunden, dass zufällige Aufnahmen von Tabak Kallus auf Nährmedium enthält ein hohes Verhältnis von CKs, Auxine, induziert werden konnte, während zufällige Wurzeln auf ein niedriges Verhältnis2-haltigem Medium induziert werden konnte. Seit dieser Feststellung hat Gewebekultur für die Ausbreitung der wirtschaftlich bedeutenden Kulturen und für die Regeneration von transgenen Pflanzen3verbreitet. Zufällige Triebe können von Gewebe als Shooting Sprossapikalmeristem Meristem, wie Blätter, Wurzeln und Internodien induziert werden. Phytohormon Behandlung ist erforderlich für die Induktion von zufällige Aufnahmen in den meisten Pflanzenarten. Allerdings unterscheiden sich die optimale Konzentrationen und Kombinationen nach Arten und unter Gewebe als Explantaten verwendet. So geht viel Aufwand in der Bestimmung der optimalen Konzentrationen und Kombinationen von Phytohormonen für Experimente.
Carapichea ipecacuanha (Brot.) L. Andersson (Ipecac) ist eine Heilpflanze, die Alkaloide wie Emetine und Cephaeline, hauptsächlich in den Wurzeln4enthält. Wurzelextrakten dienen als schleimlösend, ein Brechmittel und eine Amoebicide5. Obwohl Ipecac natürlich in den tropischen Regenwäldern von Brasilien wächst, es ungern Samen in Kultur gesetzt, und die Keimrate verringert während der Lagerung der Samen in Japan mit seinen kälteren Klima6. Stattdessen ist es durch Gewebekultur fortgepflanzt, in denen zufällig schießen Formation auf Internodien ist die effizienteste Methode7,8. Interessanterweise können zufällige schießt in dieser Spezies ohne Phytohormon Behandlung8induziert werden.
Zufällige Triebe bilden sich auf der Epidermis in der apikalen Region der Internodien Segmente ohne callusing, aber nicht in den basalen Region9. Dieser Unterschied zeigt Gewebe Polarität in der Internodien Segmente, die wahrscheinlich unter phytohormonal Regelung. Das Ipecac-Kultur-System ermöglicht eine einzigartige Gelegenheit, Änderungen im endogenen Phytohormon Ebenen während zufällig schießen Bildung zu analysieren. Hier stellen wir unsere Methode für die Analyse der endogenen Ebenen ein Auxin (Indol-3-acetic Acid (IAA)) und vier CKs (Isopentenyl Adenin (iP), Isopentenyl Adenin Riboside (iPR), Trans-Zeatin (tZ) und Trans-Zeatin Riboside (tZR)) in Internodien Segmente durch den Einsatz von LC-MS/MS.

<table><tbody><tr><td>[2H5]indole-3-acetic acid</td><td>Olchemlm Ltd</td><td>031 1531</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>[2H5]trans-zeatin</td><td>Olchemlm Ltd</td><td>030 0301</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>[2H5]trans-zeatin riboside</td><td>Olchemlm Ltd</td><td>030 0311</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>[2H6]N6-isopentenyl adenine</td><td>Olchemlm Ltd</td><td>030 0161</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>[2H6]N6-isopentenyl adenosine</td><td>Olchemlm Ltd</td><td>030 0171</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>indole-3-acetic acid</td><td>Wako</td><td>098 00181</td><td>standard for LC-MS/MS</td></tr><tr><td>trans-zeatin</td><td>SIGMA-ALDRICH</td><td>Z0876 5MG</td><td>standard for LC-MS/MS</td></tr><tr><td>trans-zeatin riboside</td><td>Wako</td><td>262 01081</td><td>standard for LC-MS/MS</td></tr><tr><td>N6-isopentenyl adenine</td><td>SIGMA-ALDRICH</td><td>D7674 1G</td><td>standard for LC-MS/MS</td></tr><tr><td>N6-isopentenyl adenosine</td><td>ACROS ORGANICS</td><td>22648 1000</td><td>standard for LC-MS/MS</td></tr><tr><td>acetonitrile hypergrade for LC-MS LiChrosolv</td><td>MERCK</td><td>1.00029.1000</td><td>solvent for LC-MS/MS</td></tr><tr><td>Water for chromatography LiChrosolv</td><td>MERCK</td><td>1.15333.1000</td><td>solvent for LC-MS/MS</td></tr><tr><td>HPLC</td><td>SHIMADZU</td><td>Prominence</td><td></td></tr><tr><td>MS</td><td>Sciex</td><td>3200QTRAP</td><td></td></tr><tr><td>Oasis HLB 30 mg/1 cc</td><td>Waters</td><td>WAT094225</td><td>cartridge column</td></tr><tr><td>Oasis MCX 30 mg/1 cc</td><td>Waters</td><td>186000252</td><td>cartridge column</td></tr><tr><td>screw neck total recovery vial</td><td>Waters</td><td>186002805</td><td></td></tr><tr><td>blue, 12 x 32mm screw neck cap and PTFE/silicone septum</td><td>Waters</td><td>186000274</td><td></td></tr><tr><td>Acquity UPLC BEH C18, 2.1x100 mm</td><td>Waters</td><td>186002350</td><td>UPLC column</td></tr><tr><td>Proshell 120 EC-C18, 2.1x50 mm</td><td>Agilent</td><td>699775-902</td><td>UPLC column</td></tr><tr><td>Digital microscope</td><td>Leica</td><td>DHS1000</td><td></td></tr><tr><td>TissueLyser II</td><td>QIAGEN</td><td>85300</td><td></td></tr><tr><td>Surgical blade</td><td>Feather</td><td>No. 22</td><td></td></tr><tr><td>Scalpel handle</td><td>Feather</td><td>No. 4</td><td></td></tr><tr><td>Savant SpeedVac/Refregerated vapor trap</td><td>Thermo Fisher Scientific</td><td>SPD111/RVT4104</td><td>vacuum concentrartor</td></tr><tr><td>Disposable glass tobe (13x100 mm)</td><td>IWAKI</td><td>9832-1310</td><td></td></tr><tr><td>Sterile petri dish</td><td>INA OPTICA</td><td>I-90-20</td><td></td></tr></tbody></table>

quantification of endogenous auxin and cytokinin during internode culture of ipecac

Zufällige Shooting Bildung ist eine wichtige Technik für die Ausbreitung der wirtschaftlich bedeutenden Kulturen und für die Regeneration von transgenen Pflanzen. Phytohormon Behandlung ist erforderlich für die Induktion von zufällige Aufnahmen bei den meisten Arten. Ob zufällig Triebe induziert werden können wird bestimmt durch das Gleichgewicht zwischen Auxin und Cytokinin (CK) Ebenen. Viel Aufwand geht in der Bestimmung der optimalen Konzentrationen und Kombinationen von Phytohormonen in jedem Gewebe als Explantaten verwendet und bei jedem Pflanzenarten. In Ipecac können jedoch zufällig Triebe auf Internodien Segmente in Kulturmedium ohne Phytohormon Behandlung induziert werden. Dies ermöglicht die inhärente Plastizität von Ipecac für Zelldifferenzierung ausgewertet werden. Um zufällige Triebe in Ipecac induzieren, kultivierten wir Internodien Segmente bei 24 ° C unter 15 µmol m−2 s−1 des Lichts in einem 14-h Licht/10-h dunklen Zyklus auf Phytohormon-freie B5 Medium verfestigt mit 0,2 % Gellan Gum für 5 Wochen. Um Phytohormon Dynamik während zufällig schießen Bildung zu untersuchen, haben wir gemessen endogenen Indol-3-acetic Acid und CKs in den Segmenten durch flüssige Chromatographie-Tandem-Massenspektrometrie LC-MS/MS. Diese Methode ermöglicht die Analyse der endogenen Indol-3-acetic Säure und CKs Ebenen auf einfache Weise. Es kann angewendet werden, um die Dynamik der endogenen Auxin und CK während der Organogenese in anderen Pflanzenarten zu untersuchen.

1St Woche hatte keine zufällige Triebe gebildet. Bei der 2Nd Woche erschienen kleine Triebe. Die Anzahl der schießt auf die 3rd und 4th Wochen erhöhte meist in den apikalen Regionen (I und II) (Abb. 2A). Inder 5 Woche die Zahl der Triebe war etwa 7 in Region ich und 5 in Region II (Abbildung 2B). Im Gegensatz dazu bildeten sich nur ein paa...

Watch this Scientific Journal Video about Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac at JoVE.com

Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac

Quantifizierung der endogenen Auxin und Cytokinin während Internodium Kultur von Ipecac

Zufällige Triebe können auf Internodien Segmente der Ipecacuanha ohne Phytohormon Behandlung induziert werden. Um Phytohormon Dynamik während zufällig schießen Bildung zu bewerten, haben wir endogenen Auxin und Cytokinin in Internodien Segmenten von LC-MS/MS gemessen.

Adventitious shoot formation is an important technique for the propagation of economically important crops and for the regeneration of transgenic plants. ...

quantification-endogenous-auxin-cytokinin-during-internode-culture

Universidad de Cartagena UDEC

Research

JoVE Journal

Environment

9.0K Aufrufe.  Toyo University. Zufällige Triebe können auf Internodien Segmente der Ipecacuanha ohne Phytohormon Behandlung induziert werden. Um Phytohormon Dynamik während zufällig schießen Bildung zu bewerten, haben wir endogenen Auxin und Cytokinin in Internodien Segmenten von LC-MS/MS gemessen.

Serie: Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac

Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ripening. Exogenous ethylene applied during early plant development triggers the triple response phenotype; a shorter and thicker hypocotyl with an exaggerated apical hook. Despite the intimate relationship between plants and bacteria, the effect of exogenous ethylene on bacteria has been greatly overlooked. This is partly due to the difficulty of controlling gaseous ethylene within the laboratory without specialized equipment. 2-Chloroethylphosphonic acid (CEPA) is a compound that decomposes into ethylene, chlorine, and phosphate in a 1:1:1:1 molar ratio when dissolved in an aqueous medium of pH 3.5 or greater. Here we describe the use of CEPA to produce in situ ethylene for the investigation of ethylene response in bacteria using the fruit-associated, cellulose-producing bacterium Komagataeibacter xylinus as a model organism. The protocols described herein include both the verification of ethylene production from CEPA via the Arabidopsis thaliana triple response assay and the effects of exogenous ethylene on K. xylinus cellulose production, pellicle properties and colonial morphology. These protocols can be adapted to examine the effect of ethylene on other microbes using appropriate growth media and phenotype analyses. The use of CEPA provides researchers with a simple and efficient alternative to pure ethylene gas for the routine determination of bacterial ethylene response.

The protocols outlined herein facilitate the convenient investigation of bacterial ethylene responses by utilizing 2-chloroethylphosphonic acid (CEPA). Ethylene is produced in situ through the decomposition of CEPA in an aqueous bacterial growth medium, circumventing the requirement for pure ethylene gas.

Unter Verwendung der Ethylen-freisetzenden Verbindung, 2-Chlorethylphosphonsäure, als Werkzeug Ethylen-Antwort in Bakterien zu studieren

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ...

Forschung

Biochemie

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an Agrobacterium rhizogenes mediated transformation system on pigeon pea. The stem is infected with A. rhizogenes carrying a binary vector, which induced callus after 7 days and adventitious roots 14 days later. The generated transgenic hairy root was identified by morphological analysis and a GFP reporter gene.To further illustrate the application range of this system, CcCIPK14 (Calcineurin B-like protein-interacting protein kinases) was transformed into pigeon pea using this transformation method. The transgenic plants were treated with jasmonic acid (JA) and abscisic acid (ABA), respectively, for the purpose of testing whether CcCIPK14 responds to those hormones. The results demonstrated that (1) exogenous hormones could significantly upregulate the expression levelof CcCIPK14, especially in CcCIPK14 over-expression (OE) plants; (2) the content of Genistein in CcCIPK14-OE lines was significantly higher than the control; (3) the expression level of two downstream key flavonoid synthase genes, CcHIDH1 and CcHIDH2, were up-regulated in the CcCIPK14-OE lines; and (4) the hairy root transgenic system can be used to study metabolically functional genes in non-model plants.

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

Here we present an efficient and stable transformation system for the functional analysis of the CcCIPK14 gene as an example, providing a technical basis for studying the metabolism of non-model plants.

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an ...

Genetik

Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC

The phosphate esters of myo-inositol, also termed inositol phosphates (InsPs), are a class of cellular regulators playing important roles in plant physiology. Due to their negative charge, low abundance and susceptibility to hydrolytic activities, the detection and quantification of these molecules is challenging. This is particularly the case for highly phosphorylated forms containing &#8216;high-energy&#8217; diphospho bonds, also termed inositol pyrophosphates (PP-InsPs). Due to its high sensitivity, strong anion exchange high-performance liquid chromatography (SAX-HPLC) of plants labeled with [3H]-myo-inositol is currently the method of choice to analyze these molecules. By using [3H]-myo-inositol to radiolabel plant seedlings, various InsP species including several non-enantiomeric isomers can be detected and discriminated with high sensitivity. Here, the setup of a suitable SAX-HPLC system is described, as well as the complete workflow from plant cultivation, radiolabeling and InsP extraction to the SAX-HPLC run and subsequent data analysis. The protocol presented here allows the discrimination and quantification of various InsP species, including several non-enantiomeric isomers and of the PP-InsPs, InsP7 and InsP8, and can be easily adapted to other plant species. As examples, SAX-HPLC analyses of Arabidopsis thaliana and Lotus japonicus seedlings are performed and complete InsP profiles are presented and discussed. The method described here represents a promising tool to better understand the biological roles of InsPs in plants.

Here we describe strong anion exchange high-performance liquid chromatography of [3H]-myo-inositol-labeled seedlings which is a highly sensitive method to detect and quantify inositol polyphosphates in plants.

Extraktion und Quantifizierung von löslichen, radioaktiv markierten Inositolpolyphosphaten aus verschiedenen Pflanzenarten mit SAX-HPLC

The phosphate esters of myo-inositol, also termed inositol phosphates (InsPs), are a class of cellular regulators playing important roles in plant ...

Tuning Degradation to Achieve Specific and Efficient Protein Depletion

The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting them for degradation. This system is exploited in many non-plant eukaryotes, such that a target protein, tagged with the AID motif, is degraded upon auxin addition. The level of TIR1 expression is critical; excessive expression leads to degradation of the AID-tagged protein even in the absence of auxin, whereas low expression leads to slow depletion. A &#946;-estradiol-inducible AID system was created, with expression of TIR1 under the control of a &#946;-estradiol inducible promoter. The level of TIR1 is tunable by changing the time of incubation with &#946;-estradiol before auxin addition. This protocol describes how to rapidly deplete a target protein using the AID system. The appropriate &#946;-estradiol incubation time depends on the abundance of the target protein. Therefore, efficient depletion depends on optimal timing that also minimizes auxin-independent depletion.

Here, we present a protocol to effectively and specifically deplete a protein of interest in the yeast Saccharomyces cerevisiae using the &#946;-est AID system.

Tuning Degradation, um eine spezifische und effiziente Proteinerschöpfung zu erreichen

The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting ...

Immunologie und Infektion

Lateral Root Inducible System in Arabidopsis and Maize

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

Lateral Root Inducible System in Arabidopsis and Maize

The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

Seitenwurzel induzierbaren Systems in<em&gt; Arabidopsis</em&gt; Und Mais

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is ...

Biologie

A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The establishment of an auxin gradient is a key event leading to phototropism and gravitropism. Previously, polar auxin transport (PAT) was shown to establish an auxin gradient in different cellular domains of plants. However, Han et al. recently demonstrated that for proper auxin gradient formation, plasmodesmal callose-mediated symplasmic connectivity between the adjacent cells is also a critical factor. In this manuscript, the strategy to elucidate the role of particular genes, which can affect phototropism and gravitropism by altering the symplasmic connectivity through modulating plasmodesmal callose synthesis, is discussed. The first step is to screen aberrant tropic responses from 3-day-old etiolated seedlings of mutants or over-expression lines of a gene along with the wild type. This preliminary screening can lead to the identification of a range of genes functioning in PAT or controlling symplasmic connectivity. The second screening involves the sorting of candidates that show altered tropic responses by affecting symplasmic connectivity. To address such candidates, the movement of a symplasmic tracer and the deposition of plasmodesmal callose were examined. This strategy would be useful to explore new candidate genes that can regulate symplasmic connectivity directly or indirectly during tropic responses and other developmental processes.

This article describes the methods for screening the genes controlling plasmodesmal permeability and hence auxin gradient during tropic response. This includes the measurement of the degree of tropic response in hypocotyl of Arabidopsis thaliana and checking plasmodesmal permeability by 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) loading and finally callose level assessment.

Eine Strategie zur Validierung der Rolle der Kallose-vermittelten Plasmodesmal Gating im Tropic Antwort

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The ...

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various &#34;omics&#34; analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e.&#160;microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

Ein Hydroponisches Co-Kultivierungssystem zur gleichzeitigen und systematischen Analyse von Pflanzen- / Mikroben-Molecular-Wechselwirkungen und Signalgebung

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

Messung der Endoreduplikation durch Durchflusszytometrie von isolierten Knolle Protoplasten

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant productivity and survival. As such, understanding the mechanisms by which stomata develop and pattern has tremendous agronomic value. This paper describes two phenotypic methods using Arabidopsis cotyledons that can be used to characterize the genes controlling stomatal development and patterning. Presented first are procedures for analyzing the stomatal phenotypes using toluidine blue O-stained cotyledons. This method is fast and reliable and does not require the use of epidermal peels, which are widely used for phenotypic analyses but require specialized training. Due to the presence of multiple cysteine residues, the identification and generation of bioactive EPF peptides that have a role in stomatal development have been challenging. Thus, presented second is a procedure used to identify stomatal ligands and monitor their biological activity by bioassays. The main advantage of this method is that it produces reproducible data relatively easily while reducing the amount of peptide solution and the time required to characterize the role of the peptides in controlling stomatal patterning and development. Overall, these well-designed protocols enhance the efficiency of studying the potential stomatal regulators, including cysteine-rich secretory peptides, which require highly complex structures for their activity.

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

This paper describes two phenotyping methods without the use of epidermal peels to characterize the genes controlling stomatal development. The first method demonstrates how to analyze a stomatal phenotype using a toluidine blue O-stained plant epidermis. The second method describes how to identify stomatal ligands and monitor their biological activities.

Identifizierung der Gene, die an der Entwicklung der Stomata beteiligt sind, mittels epidermalem Phänotyp-Scoring

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant ...

Hochauflösende Bildgebung der Chlorophyll-Biosynthese in Arabidopsis-Keimlingen

Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of Arabidopsis De-etiolation

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly dynamic process of chlorophyll biosynthesis is triggered during the shift from the dark to the light in flowering plants. At the moment when etiolated seedlings are exposed to the first traces of sunlight, rapid (in order of seconds) conversion of protochlorophyllide into chlorophyllide is mediated by unique light-accepting protein complexes, leading via subsequent metabolic steps to the production of fully functional chlorophyll. Standard techniques for chlorophyll content analysis include pigment extraction from detached plant tissues, which does not apply to studying such fast processes. To investigate chlorophyll kinetics in vivo with high accuracy and spatiotemporal resolution in the first hours after light-induced de-etiolation, an instrument and protocol were developed. Here, we present a detailed procedure designed for statistically robust quantification of chlorophyll in the early stages of Arabidopsis de-etiolation.

Autor im Rampenlicht: Nicht-invasive hochauflösende Messung der Chlorophyllsynthese während der De-Etiolation

Here, we describe an advanced tool designed for chlorophyll biosynthesis monitoring during the early stages of Arabidopsis seedling de-etiolation. The novel methodology provides non-invasive real-time chlorophyll fluorescence imaging at high spatial and temporal resolution.

Nicht-invasiver Assay zur Bestimmung der Chlorophyll-Biosynthese-Kinetik in frühen Stadien der Arabidopsis-De-etiolation

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly ...

Quantifizierung der endogenen Auxin und Cytokinin während Internodium Kultur von Ipecac

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Unter Verwendung der Ethylen-freisetzenden Verbindung, 2-Chlorethylphosphonsäure, als Werkzeug Ethylen-Antwort in Bakterien zu studieren

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

Extraktion und Quantifizierung von löslichen, radioaktiv markierten Inositolpolyphosphaten aus verschiedenen Pflanzenarten mit SAX-HPLC

Tuning Degradation, um eine spezifische und effiziente Proteinerschöpfung zu erreichen

Seitenwurzel induzierbaren Systems in

Eine Strategie zur Validierung der Rolle der Kallose-vermittelten Plasmodesmal Gating im Tropic Antwort

Ein Hydroponisches Co-Kultivierungssystem zur gleichzeitigen und systematischen Analyse von Pflanzen- / Mikroben-Molecular-Wechselwirkungen und Signalgebung

Messung der Endoreduplikation durch Durchflusszytometrie von isolierten Knolle Protoplasten

Identifizierung der Gene, die an der Entwicklung der Stomata beteiligt sind, mittels epidermalem Phänotyp-Scoring

Nicht-invasiver Assay zur Bestimmung der Chlorophyll-Biosynthese-Kinetik in frühen Stadien der Arabidopsis-De-etiolation