Name: quantification of endogenous auxin and cytokinin during internode culture of ipecac
Uploaded: 2018-03-15T14:00:00
Description: Watch this Scientific Journal Video about Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac at JoVE.com

Vi er taknemmelige for Mr. Akira Murakami af Institut for anvendt Biosciences, Toyo-universitetet og Mr. Koudai Taniguchi i Gunma landbrugs Technology Center for deres tekniske bistand. Vi er også taknemmelige til Professor Shosaku Kashiwada og Dr. Uma Maheswari Rajagopalan, Toyo Universitet for deres forslag. Denne undersøgelse var delvist understøttet af Forskningscentret for livet og miljøvidenskab, Toyo Universitet.

Bemærk: Ipecac (C. ipecacuanha) blev brugt i denne undersøgelse, fordi det letter analysen af endogene Phytohormoner.
1. vækst betingelser til at fremkalde utilsigtet skud af Ipecac
<ol><li>Forberede phytohormone-fri B5 medium justeres til pH 5.710, og tilføje 0,2% gellan tyggegummi. Steriliseres ved autoklavering.</li>
	<li>Hæld 25 mL af autoklaveres mediet i en steril petriskål (90 mm × 20 mm).</li>
	<li>Skær 8-mm internodal segmenter af ipecac plan...

<ol>
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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. 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For at identificere fordelingen af Phytohormoner involveret i organogenesis, det er vigtigt at bruge plantematerialer, hvor organogenesis kan iagttages på phytohormone-gratis medium, fordi når Phytohormoner anvendes eksogent på explants for at fremkalde skud eller rødder, de påvirker den hele eksplantat, hvilket gør det vanskeligt at vurdere de iboende plasticitet af planter i Celledifferentiering og organogenesis. Utilsigtet skud kan induceres på phytohormone-fri kultur medier i andre plantearter såsom Diant...

Gottlieb Haberlandt (1854-1945) foreslået begrebet "totipotency", af hvilke anlæg kan celler dividere, differentiere og regenerere hele planter selv efter deres forudgående differentiering af specifikke celletyper i modne planter1. I vævskultur bestemmes hvorvidt planten regeneration kan være fremkaldt eller ikke af den kombination og koncentration af eksogent anvendt Phytohormoner i vækstmediet. Skoog og Miller fandt, at utilsigtet skud kunne være induceret fra tobak callus kultur medium, der indeholder en høj andel af CKs til auxins, der henviser til, at utilsigtet rødder kunne være fremkaldt på medium, der indeholder en lav ratio2. Siden denne konstatering, har væv kultur været udbredt formeringsmateriale af økonomisk vigtige afgrøder og til regenerering af transgene planter3. Utilsigtet skud kan være fremkaldt af væv end skyde yderste meristem, som blade, rødder og stængelled. Phytohormone behandling er påkrævet for induktion af utilsigtet skud i de fleste plantearter. Optimal koncentrationer og kombinationer varierer dog af arter og blandt væv bruges som explants. Således, meget indsats går til bestemmelse af optimal koncentrationer og kombinationer af Phytohormoner for eksperimenter.
Carapichea ipecacuanha (Brot.) L. Andersson (ipecac) er en lægeplante, der indeholder alkaloider såsom emetine og cephaeline, hovedsagelig i rødder4. Rod ekstrakter anvendes som en slimløsende, et brækmiddel og en amoebicide5. Selvom ipecac vokser naturligt i tropiske regnskove i Brasilien, det er tilbageholdende med at sætte frø i kultur og spiring sats falder under frø opbevaring i Japan, med sit koldere klima6. I stedet, det er formeret af vævskultur, som utilsigtet skyde dannelse på stængelled er den mest effektive metode7,8. Interessant, kan utilsigtet skud være fremkaldt i denne arter uden phytohormone behandling8.
Utilsigtet skud er dannet på epidermis i den apikale region internodal segmenter uden callusing, men ikke i basal region9. Denne forskel angiver væv polaritet i internodal segmenter, som er formentlig under phytohormonal forordning. Ipecac kultur system giver en enestående mulighed for at analysere ændringer i endogene phytohormone niveauer under utilsigtet skyde dannelse. Her vil vi præsentere vores metode til analyse af de endogene niveauer af én auxin (indol-3-eddikesyre (IAA)) og fire CKs (isopentenyl adenin (iP), isopentenyl adenin riboside (iPR), trans-zeatin (tZ), og trans-zeatin riboside (tZR)) i internodal segmenter ved hjælp af LC-MS/MS.

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		<col />
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		<col />
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">[2H5]indole-3-acetic acid</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">031 1531</td>
			<td style="width:167px;">Internal standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">[2H5]trans-zeatin</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">030 0301</td>
			<td>Internal standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">[2H5]trans-zeatin riboside</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">030 0311</td>
			<td>Internal standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">[2H6]N6-isopentenyl adenine</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">030 0161</td>
			<td>Internal standard for LC-MS/MS</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">[2H6]N6-isopentenyl adenosine</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">030 0171</td>
			<td>Internal standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">indole-3-acetic acid</td>
			<td style="width:176px;">Wako</td>
			<td>098 00181</td>
			<td>standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">trans-zeatin</td>
			<td style="width:176px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">Z0876 5MG</td>
			<td>standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">trans-zeatin riboside</td>
			<td style="width:176px;">Wako</td>
			<td>262 01081</td>
			<td>standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N6-isopentenyl adenine</td>
			<td style="width:176px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">D7674 1G</td>
			<td>standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N6-isopentenyl adenosine</td>
			<td style="width:176px;">ACROS ORGANICS</td>
			<td style="width:119px;">22648 1000</td>
			<td>standard for LC-MS/MS</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">acetonitrile hypergrade for LC-MS LiChrosolv</td>
			<td style="width:176px;">MERCK</td>
			<td style="width:119px;">1.00029.1000</td>
			<td>solvent for LC-MS/MS</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Water for chromatography LiChrosolv</td>
			<td style="width:176px;">MERCK</td>
			<td style="width:119px;">1.15333.1000</td>
			<td>solvent for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HPLC</td>
			<td style="width:176px;">SHIMADZU</td>
			<td style="width:119px;">Prominence</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MS</td>
			<td style="width:176px;">Sciex</td>
			<td style="width:119px;">3200QTRAP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oasis HLB 30 mg/1 cc</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">WAT094225</td>
			<td>cartridge column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oasis MCX 30 mg/1 cc</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">186000252</td>
			<td>cartridge column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">screw neck total recovery vial</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">186002805</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">blue, 12 x 32mm screw neck cap and PTFE/silicone septum</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">186000274</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Acquity UPLC BEH C18, 2.1x100 mm</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">186002350</td>
			<td>UPLC column</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Proshell 120 EC-C18, 2.1x50 mm</td>
			<td style="width:176px;">Agilent</td>
			<td style="width:119px;">699775-902</td>
			<td>UPLC column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Digital microscope</td>
			<td style="width:176px;">Leica</td>
			<td style="width:119px;">DHS1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TissueLyser II</td>
			<td style="width:176px;">QIAGEN</td>
			<td style="width:119px;">85300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical blade</td>
			<td style="width:176px;">Feather</td>
			<td style="width:119px;">No. 22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scalpel handle</td>
			<td style="width:176px;">Feather</td>
			<td style="width:119px;">No. 4</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Savant SpeedVac/Refregerated vapor trap</td>
			<td style="width:176px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">SPD111/RVT4104</td>
			<td>vacuum concentrartor</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Disposable glass tobe (13x100 mm)</td>
			<td style="width:176px;">IWAKI</td>
			<td style="width:119px;">9832-1310</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile petri dish</td>
			<td style="width:176px;">INA OPTICA</td>
			<td style="width:119px;">I-90-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of endogenous auxin and cytokinin during internode culture of ipecac

Utilsigtet skyde dannelse er en vigtig teknik for udbredelsen af økonomisk vigtige afgrøder og regenerering af transgene planter. Phytohormone behandling er påkrævet for induktion af utilsigtet skud i de fleste arter. Om utilsigtet skud kan induceres bestemmes af balancen mellem auxin og cytokinin (CK) niveau. Stor indsats går til bestemmelse af optimal koncentrationer og kombinationer af Phytohormoner i hver væv bruges som explants og i enkelte plantearter. I ipecac, imidlertid kan utilsigtet skud induceres på internodal segmenter i dyrkningsmediet uden phytohormone behandling. Dette giver mulighed for den iboende plasticitet af ipecac celle differentiering skal evalueres. For at fremkalde utilsigtet skud i ipecac, kulturperler vi internodal segmenter på 24 ° C under 15 µmol m−2 s1 af lys i en 14-h lys/10-h mørke cyklus på phytohormone-gratis B5 medium solidificeret med 0,2% gellan tyggegummi i 5 uger. For at undersøge phytohormone dynamics under utilsigtet skyde dannelse, målte vi endogene indol-3-eddikesyre og CKs i segmenter af liquid chromatography-tandem massespektrometri LC-MS/MS. Denne metode giver mulighed for analyse af endogene indol-3-eddikesyre og CKs niveauer på en enkel måde. Det kan anvendes til at undersøge dynamics af endogene auxin og CK under organogenesis i andre plantearter.

1st uge, havde ingen utilsigtet skud dannet. Uge 2nd små skud dukkede op. På de 3rd og 4th uger, antallet af skyder øget mest i de apikale regioner (I og II) (figur 2A). 5th uge, antallet af skud var ca 7 i regionen og 5 i region II (figur 2B). I modsætning, blev kun et par skud dannet i region III og IV.
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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

Kvantificering af endogene Auxin og Cytokinin under internodium kultur af Ipecac

Utilsigtet skud kan induceres på internodal segmenter af ipecac uden phytohormone behandling. For at vurdere phytohormone dynamics under utilsigtet skyde dannelse, målte vi endogene auxin og cytokinin i internodal segmenter af LC-MS/MS.

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

The overall goal of this procedure is to describe how endogenous phytohormone levels change during adventitious shoot formation in ipecac. This method can help answer key questions related to tissue regeneration in the plant tissue culture wheel. The main advantage of this technique is the endogenous level of Indole-3-acetic acid and cytokinines can be simply analyzed with high sensitivity.The implication of this technique extend throughout establishment of plant tissue degeneration because changes in endogenous phytohormone level trigger adventitious shoot formation. To begin, prepare a phytohormone-free B5 medium and add 0.2 percent gellan gum. Then, autoclave the medium to sterilize it.Using a surgical scalpel on a sterile acrylic plate, slice eight millimeter internodal segments of ipecac plantlets and place them on the medium. Then, culture the ipecac segments at 24 degrees Celsius under 15 micromole per square meter seconds of light in a 14-hour light, 10-hour dark cycle for five weeks. After culturing the ipecac segments, identify regions one through four in each segment.Once a week, use a microscope to count the number of adventitious shoots in each region. First, place a five millimeter zirconia bead into four two-milliliter sample tubes. Then, use a surgical scalpel on a sterile acrylic plate to cut the ipecac segments into regions I through IV.Then collect eight segments from each region into one sample tube.Weigh each of the samples and freeze them in liquid nitrogen. Next, use a bead-based homogenizer to crush the frozen samples. Use a vortex mixer to suspend the samples in one milliliter of the acetonitrile with 500 picograms of each internal standard of oxen and CKs.Home the samples at four degrees Celsius for one hour. And then centrifuge the samples at 3500 G for five minutes at room temperature. Wash the pellet in 80 percent acetal nitrile with one percent acetic acid.Then repeat the centrifugation and use a disposable class tube to combine the supernatants. Add 600 microliters of water with one percent acetic acid to the combined supernatant. Then use a vacuum concentrator to evaporate the acetal nitrile.Load the sample solutions into individual equilibrated HLB cartridges. And wash the cartridges with one milliliter of water with one percent acetic acid. Then allude all of the hormones with two milliliters of 80 percent acetyl nitrile with one percent acetic acid in a glass tube.Using a vacuum concentrator, evaporate the acetal nitrile to obtain extract in water with one percent acetic acid. Load the sample solutions into individual equilibrated MCX cartridges and wash the cartridges with one milliliter of water with one percent acetic acid. Then allude IAA in a glass tube, with two milliliters of 30 percent acetal nitrile with one percent acetic acid.Then wash the cartridges with two milliliters of 80 percent acetal nitrile with one percent acetic acid. Next, wash the cartridges with two milliliters of water and one milliliter of water with five percent aqueous ammonia in a draft chamber. Allude the CKs in a glass tube with two milliliters of 60 percent acetal nitrile with five percent aqueous ammonia in a draft chamber.Then use a vacuum concentrator to evaporate the solvent of each hormone fraction. Stir the samples at minus thirty degrees Celsius until LCMSMS analysis. First, dissolve each hormone extract in a tube with 600 microliters of methanol.Then transfer the solution to a screw-neck total recovery vial and use a vacuum concentrator to evaporate the solvent. Dissolve the IAA fraction in 20 microliters of 30 percent acetal nitrile and the CK fractions in 20 microliters of water with one percent acetic acid. Next use a triple quadrupole MS system equipped with an HPLC system to analyze the samples in positive ion mode.Finally quantify endogenous IAA in CK levels against a standard curve of the ration of unlabeled to deuterium label standards. In this protocol endogenous oxidant cytokinin in adventitious chutes was measured via LCMSMS for five weeks. At the first week, no adventitious chutes had formed and the first small chutes appeared during the second week.During the third and fourth weeks, the number of chutes increased mostly in regents one and two. During the fifth week the number of chutes was approximately seven in region one, and five in region two. Before culture, the IAA level was slightly higher in region one than in regions two, three, and four.During the first week, the IAA level increased greatly in region four, and decreased slightly in regions one, two, and three. By five weeks of culture an IAA concentration gradient emerged. With levels increasing from region one to region four.Before culture, there were only trace levels of most of the CKs. At the first week, the tZR level increased to 13.8 picograms per milligram in region two. And 18.1 picograms per milligram in region three.In contrast the levels of tZ, iP and iPR changed only slightly during culture. While attempting this procedure it's important to remember that endogenous part hormone are stable. After it's developed man, this technique paves the way for researchers in the field of plant tissue culture.To explore adventitious formation in other organisms. After watching this video, you should have a good understanding of how to analyze endogenous Indole-3-acetic acid and cytokinines.

Research

JoVE Journal

Environment

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615. Måling af Enterovirus og norovirus Forekomst i vand efter kultur og RT-qPCR. I. Indsamling af virus prøver

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae

Kvantificering af tungmetaller og andre uorganiske forureninger på produktivitet mikroalger

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages ...

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Samtidig DNA-RNA-ekstraktion fra kystsedimenter og kvantificering af 16S rRNA-generne og udskrifter af Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

Capturing Flow-weighted Water and Suspended Particulates from Agricultural Canals During Drainage Events

Indfange Flow-vægtede vand og svaevestoev fra landbruget kanaler under dræning begivenheder

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage ...

The Effect of the Application of Thyme Essential Oil on Microbial Load During Meat Drying

Effekten af anvendelsen af timian æteriske olie på mikrobielle belastning under kød tørring

Meat is a high protein meal that is used in the preparation of jerky, a popular food snack, where preservation and safety are important. To assure food ...

Stress Distribution During Cold Compression of Rocks and Mineral Aggregates Using Synchrotron-based X-Ray Diffraction

Stress Distribution under kolde komprimering af klipper og mineralske aggregater ved hjælp af synkrotron-baserede røntgen diffraktion

We report detailed procedures for performing compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus (D-DIA) ...

Chemical Isolation, Quantification, and Separation of Skin Lipids from Reptiles

Kemiske Isolation, kvantificering og adskillelse af huden lipider fra krybdyr

Reptiles signal to conspecifics using lipids in their skin, primarily to enable mate tracking and assessment. The isolation of these lipids has utility in ...

Split Point Analysis and Uncertainty Quantification of Thermal-Optical Organic/Elemental Carbon Measurements

Analyse af Split punkt og usikkerheds kvantificering af termisk-optiske organiske/Elementale kulstof målinger

Researchers from myriad fields frequently seek to quantify and classify concentrations of carbonaceous aerosols as organic carbon (OC) or elemental carbon ...

Processing Embryo, Eggshell, and Fungal Culture for Scanning Electron Microscopy

Behandling af embryoer, æggeskaller og svampe kultur til scanning af elektronmikroskopi

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, ...