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

We are grateful to Mr. Akira Murakami of the Department of Applied Biosciences, Toyo University, and Mr. Koudai Taniguchi of the Gunma Agricultural Technology Center for their technical assistance. We are also grateful to Professor Shosaku Kashiwada and Dr. Uma Maheswari Rajagopalan, Toyo University for their suggestions. This study was supported in part by the Research Center for Life and Environmental Sciences, Toyo University.

Note: Ipecac (C. ipecacuanha) was used in this study because it facilitates the analysis of endogenous phytohormones.
1. Growth Conditions to Induce Adventitious Shoots of Ipecac
<ol>
	<li>Prepare phytohormone-free B5 medium adjusted to pH 5.710, and add 0.2% gellan gum. Sterilize by autoclaving.</li>
	<li>Pour 25 mL of the autoclaved medium into a sterile Petri dish (90 mm &#215; 20 mm).</li>
	<li>Cut 8-mm internodal segments of ipecac plantlets using a surg...

<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>

To identify the distribution of phytohormones involved in organogenesis, it is important to use plant materials in which organogenesis can be observed on phytohormone-free medium, because when phytohormones are exogenously applied to explants for inducing shoots or roots, they affect the whole explant, making it difficult to evaluate the inherent plasticity of plants in cell differentiation and organogenesis. Adventitious shoots can be induced on phytohormone-free culture media in other plant species such as Dianthus...

Gottlieb Haberlandt (1854-1945) proposed the concept of &#34;totipotency&#34;, by which plant cells can divide, differentiate, and regenerate whole plants even after their prior differentiation into specific cell types in mature plants1. In tissue culture, whether plant regeneration can be induced or not is determined by the combination and concentration of exogenously applied phytohormones in the growth medium. Skoog and Miller found that adventitious shoots could be induced from tobacco callus on culture medium containing a high ratio of CKs to auxins, whereas adventitious roots could be induced on medium containing a low ratio2. Since that finding, tissue culture has been widely used for the propagation of economically important crops and for the regeneration of transgenic plants3. Adventitious shoots can be induced from tissues other than shoot apical meristem, such as leaves, roots, and internodes. Phytohormone treatment is required for the induction of adventitious shoots in most plant species. However, the optimum concentrations and combinations differ by species and among tissues used as explants. Thus, much effort goes into determining the optimum concentrations and combinations of phytohormones for experiments.
Carapichea ipecacuanha (Brot.) L. Andersson (ipecac) is a medicinal plant that contains alkaloids such as emetine and cephaeline, mainly in the roots4. Root extracts are used as an expectorant, an emetic, and an amoebicide5. Although ipecac grows naturally in the tropical rainforests of Brazil, it is reluctant to set seeds in culture, and the germination rate decreases during seed storage in Japan, with its colder climate6. Instead, it is propagated by tissue culture, in which adventitious shoot formation on internodes is the most efficient method7,8. Interestingly, adventitious shoots can be induced in this species without phytohormone treatment8.
Adventitious shoots are formed on the epidermis in the apical region of internodal segments without callusing, but not in the basal region9. This difference indicates tissue polarity in internodal segments, which is probably under phytohormonal regulation. The ipecac culture system allows a unique opportunity to analyze changes in endogenous phytohormone levels during adventitious shoot formation. Here we introduce our method for the analysis of the endogenous levels of one auxin (indole-3-acetic acid (IAA)) and four CKs (isopentenyl adenine (iP), isopentenyl adenine riboside (iPR), trans-zeatin (tZ), and trans-zeatin riboside (tZR)) in internodal segments through the use of LC-MS/MS.

<table border="0" cellpadding="0" cellspacing="0" style="width:677px;" width="678">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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

Adventitious shoot formation is an important technique for the propagation of economically important crops and for the regeneration of transgenic plants. Phytohormone treatment is required for the induction of adventitious shoots in most species. Whether adventitious shoots can be induced is determined by the balance between auxin and cytokinin (CK) levels. Much effort goes into determining optimum concentrations and combinations of phytohormones in each tissue used as explants and in each plant species. In ipecac, however, adventitious shoots can be induced on internodal segments in culture medium without phytohormone treatment. This allows the inherent plasticity of ipecac for cell differentiation to be evaluated. To induce adventitious shoots in ipecac, we cultured internodal segments at 24 &#176;C under 15 &#181;mol m&#8722;2 s&#8722;1 of light in a 14-h light/10-h dark cycle on phytohormone-free B5 medium solidified with 0.2% gellan gum for 5 weeks. To investigate phytohormone dynamics during adventitious shoot formation, we measured endogenous indole-3-acetic acid and CKs in the segments by liquid chromatography-tandem mass spectrometry LC-MS/MS. This method allows analysis of endogenous indole-3-acetic acid and CKs levels in a simple manner. It can be applied to investigate the dynamics of endogenous auxin and CK during organogenesis in other plant species.

At the 1st week, no adventitious shoots had formed. At the 2nd week, small shoots appeared. At the 3rd and 4th weeks, the number of shoots increased mostly in the apical regions (I and II) (Figure 2A). At the 5th week, the number of shoots was approximately 7 in region I and 5 in region II (Figure 2B). In contrast, only a few shoots were form...

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

Adventitious shoots can be induced on internodal segments of ipecac without phytohormone treatment. To evaluate phytohormone dynamics during adventitious shoot formation, we measured endogenous auxin and cytokinin in internodal segments by 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.

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

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 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

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

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages include high potential yield, use of non-arable land and integration with waste streams. The nutrient requirements of a large-scale microalgae production system will require the coupling of cultivation systems with industrial waste resources, such as carbon dioxide from flue gas and nutrients from wastewater. Inorganic contaminants present in these wastes can potentially lead to bioaccumulation in microalgal biomass negatively impact productivity and limiting end use. This study focuses on the experimental evaluation of the impact and the fate of 14 inorganic contaminants (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Se, Sn, V and Zn) on Nannochloropsis salina growth. Microalgae were cultivated in photobioreactors illuminated at 984 &#181;mol m-2 sec-1 and maintained at pH 7 in a growth media polluted with inorganic contaminants at levels expected based on the composition found in commercial coal flue gas systems. Contaminants present in the biomass and the medium at the end of a 7 day growth period were analytically quantified through cold vapor atomic absorption spectrometry for Hg and through inductively coupled plasma mass spectrometry for As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Sb, Se, Sn, V and Zn. Results show N. salina is a sensitive strain to the multi-metal environment with a statistical decrease in biomass yieldwith the introduction of these contaminants. The techniques presented here are adequate for quantifying algal growth and determining the fate of inorganic contaminants.

Integration of microalgal cultivation with industrial flue gas will ultimately introduce heavy metals and other inorganic compounds into the growth media. This study presents a procedure used to determine the end fate and impact of heavy metals and inorganic contaminants on the growth of Nannochloropsis salina grown in photobioreactors.

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

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 taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with &#39;end-point&#39; PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.

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

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage discharge events. Farm canals can be enriched by nutrients such as phosphorus (P) that are susceptible to transport. Phosphorus in the form of suspended particulates can significantly contribute to the overall P loads in drainage water. A settling tank experiment was conducted to capture suspended particulates during discrete drainage events. Farm canal discharge water was collected in a series of two 200 L settling tanks over the entire duration of the drainage event, so as to represent a composite subsample of the water being discharged. Imhoff settling cones are ultimately used to settle out the suspended particulates. This is achieved by siphoning water from the settling tanks via the cones. The particulates are then collected for physico-chemical analyses.

Nutrients present in particulate form can contribute significantly to the overall loads in agricultural drainage waters. This study describes a novel method to capture flow-weighted water and suspended particulates from farm canal drainage over the entire duration of the drainage event.

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

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 safety and to extend the shelf life of meat and meat products, the use of either synthetic or natural preservatives have been applied to control and eliminate foodborne bacteria. A growing interest in the application of natural food additives for meat has increased. Microorganisms, such as Escherichia coli, contaminate meat and meat products, causing foodborne illnesses. Therefore, it is necessary to improve the meat conservation process. However, the use of essential oils when the meat is being dried has not been deeply studied. In this regard, there is an opportunity to increase the value of dried meat and reduce the risk of foodborne illnesses by applying essential oils during the drying process. In this protocol, we present a novel method of applying thyme essential oil (TEO) during meat drying, specifically in vapor form directly in a drying chamber. For evaluation, we use Minimal Inhibitory Concentration (MIC) to detect the number of harmful bacteria in the treated samples compared to raw samples. The preliminary results show that this method is a viable and alternative option to synthetic preservatives and that it significantly reduces microbial load in dried meat.

Microorganisms such as Escherichia coli that contaminate meat products cause foodborne illnesses. The use of essential oils in the meat drying process has not been deeply studied. Here, we present a novel method of applying thyme essential oil to meat during drying to reduce the microbial load in dried meat.

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

We report detailed procedures for performing compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus (D-DIA) coupled with synchrotron X-radiation. A cube-shaped sample assembly is prepared and compressed, at room temperature, by a set of four X-ray transparent sintered diamond anvils and two tungsten carbide anvils, in the lateral and the vertical planes, respectively. All six anvils are housed within a 250-ton hydraulic press and driven inward simultaneously by two wedged guide blocks. A horizontal energy dispersive X-ray beam is projected through and diffracted by the sample assembly. The beam is commonly in the mode of either white or monochromatic X-ray. In the case of white X-ray, the diffracted X-rays are detected by a solid-state detector array that collects the resulting energy dispersive diffraction pattern. In the case of monochromatic X-ray, the diffracted pattern is recorded using a two-dimensional (2-D) detector, such as an imaging plate or a charge-coupled device (CCD) detector. The 2-D diffraction patterns are analyzed to derive lattice spacings. The elastic strains of the sample are derived from the atomic lattice spacing within grains. The stress is then calculated using the predetermined elastic modulus and the elastic strain. Furthermore, the stress distribution in two-dimensions allow for understanding how stress is distributed in different orientations. In addition, a scintillator in the X-ray path yields a visible light image of the sample environment, which allows for the precise measurement of sample length changes during the experiment, yielding a direct measurement of volume strain on the sample. This type of experiment can quantify the stress distribution within geomaterials, which can ultimately shed light on the mechanism responsible for compaction. Such knowledge has the potential to significantly improve our understanding of key processes in rock mechanics, geotechnical engineering, mineral physics, and material science applications where compactive processes are important.

We report detailed procedures for compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus coupled with synchrotron X-radiation. Such experiments allow quantification of the stress distribution within samples, that ultimately sheds light on compaction processes in geomaterials.

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

Reptiles signal to conspecifics using lipids in their skin, primarily to enable mate tracking and assessment. The isolation of these lipids has utility in research focused on evolutionary patterns and mechanisms of chemical communication, in addition to understanding the waterproofing role of lipids in the evolution of terrestrial life. In an applied approach, such skin-based cues have potential use for wildlife managers dealing with invasive species. The main steps for quantifying reptile skin lipids in the protocol presented here include extraction, total lipid determination, and fractionation via column chromatography, the latter process resulting in purified eluates of compounds which can then either be analyzed to assign compound identifications (e.g., gas chromatography-mass spectrometry [GC-MS]) and/or used directly in more refined bioassays. Skin lipids can be extracted from living skin, shed skin, or dead whole animals, using nonpolar organic solvents (e.g., hexane, benzene, toluene). Extraction solubilizes the lipids and, then, the solvent can be evaporated to yield a measurable lipid-only extract. Fractionation involves the separation of the total lipid extract into specific eluates via traditional column chromatography. The total lipid extract is first bound to a substrate-based column (e.g., alumina) and, then, individual eluates (&#34;fractions&#34;) of solvent at specific volumes are passed sequentially through the column to elute sets of compounds from the lipid mixture based on common polarity. The fractions progress in polarity at a standardized sequence by increasing the relative amount of polar solvent (e.g., diethyl ether) in nonpolar solvent. In this manuscript, we describe several methods for extracting skin lipids of reptiles and, then, provide a standard protocol for isolating different sets of compounds based on polarity, using traditional column chromatography. Whole lipid extracts or specific fractions can, then, be used in bioassays to determine any biological activity elicited by the compounds therein.

In reptiles, skin lipids from conspecifics are crucial for sexual signaling, with potential use in invasive species management. Here, we describe protocols for extracting skin lipids from shed skin or whole animals, determining and analyzing the total lipid mass, and separating the lipids using fractionation via column chromatography.

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

Researchers from myriad fields frequently seek to quantify and classify concentrations of carbonaceous aerosols as organic carbon (OC) or elemental carbon (EC). This is commonly accomplished using thermal-optical OC/EC analyzers (TOAs), which enable measurement via controlled thermal pyrolysis and oxidation under specific temperature protocols and within constrained atmospheres. Several commercial TOAs exist, including a semi-continuous instrument that enables on-line analyses in the field. This instrument employs an in-test calibration procedure that requires relatively frequent calibration. This article details a calibration protocol for this semi-continuous TOA and presents an open-source software tool for data analysis and rigorous Monte Carlo quantification of uncertainties. Notably, the software tool includes novel means to correct for instrument drift and identify and quantify the uncertainty in the OC/EC split point. This is a significant improvement on the uncertainty estimation in the manufacturer&#8217;s software, which ignores split point uncertainty and otherwise uses fixed equations for relative and absolute errors (generally leading to under-estimated uncertainties and often yielding non-physical results as demonstrated in several example data sets). The demonstrated calibration protocol and new software tool enabling accurate quantification of combined uncertainties from calibration, repeatability, and OC/EC split point are shared with the intent of assisting other researchers in achieving better measurements of OC, EC, and total carbon mass in aerosol samples.

This article presents a protocol and software tool for the quantification of uncertainties in the calibration and data analysis of a semi-continuous thermal-optical organic/elemental carbon analyzer.

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

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, methods involved in processing different biological samples involve unique practices. All conventional practices described in the literature for processing samples still find useful applications, but subtle changes in the sample preparation can alter image quality, as well as, introduce artifacts. Hence, using a unique sample preparation technique specific to the type of tissue analyzed is required to obtain a good quality image with ultrastructural resolution. The focus of this study is to provide the optimal sample preparation protocols for imaging embryos, rigid eggshells, and fungal cultures using SEM. The following optimizations were recommended to yield good results for the three different delicate biological samples studied. Use of milder fixatives like 4% paraformaldehyde or 3% glutaraldehyde followed by dehydration with ethanol series is mandatory. Fungal mycelium on agar blocks obtained by slide cultures yields a better ultrastructural integrity compared to cultures taken directly from agar plates. Chemical drying of embryos with HMDS provides drying without introducing surface tension artifacts compared to critical point drying. HMDS prevents cracking caused by shrinkage as samples are less brittle during drying. However, for fungal culture, critical point drying provides acceptable image quality compared to chemical drying. Eggshells can be imaged with no special preparation steps except for thorough washing and air drying prior to mounting. Preparation methodologies were standardized based on acceptable image quality obtained with each trial.&#160;

Here, we present detailed processing protocols for imaging delicate tissue samples using scanning electron microscopy (SEM). Three different processing methods, namely, hexamethyl disilazana (HMDS) chemical drying, simple air drying, and critical point drying are described for preparing rigid eggshells, embryos at early developmental stages, and fungal cultures respectively.

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

Two-Dimensional Visualization and Quantification of Labile, Inorganic Plant Nutrients and Contaminants in Soil

We describe a method for two-dimensional (2D) visualization and quantification of the distribution of labile (i.e., reversibly adsorbed) inorganic nutrient (e.g., P, Fe, Mn) and contaminant (e.g., As, Cd, Pb) solute species in the soil adjacent to plant roots (the &#8216;rhizosphere&#8217;) at sub-millimeter (~100 &#181;m) spatial resolution. The method combines sink-based solute sampling by the diffusive gradients in thin films (DGT) technique with spatially resolved chemical analysis by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The DGT technique is based on thin hydrogels with homogeneously distributed analyte-selective binding phases. The variety of available binding phases allows for the preparation of different DGT gel types following simple gel fabrication procedures. For DGT gel deployment in the rhizosphere, plants are grown in flat, transparent growth containers (rhizotrons), which enable minimal invasive access to a soil-grown root system. After a pre-growth period, DGT gels are applied to selected regions of interest for in situ solute sampling in the rhizosphere. Afterwards, DGT gels are retrieved and prepared for subsequent chemical analysis of the bound solutes using LA-ICP-MS line-scan imaging. Application of internal normalization using 13C and external calibration using matrix-matched gel standards further allows for the quantification of the 2D solute fluxes. This method is unique in its capability to generate quantitative, sub-mm scale 2D images of multi-element solute fluxes in soil-plant environments, exceeding the achievable spatial resolution of other methods for measuring solute gradients in the rhizosphere substantially. We present the application and evaluation of the method for imaging multiple cationic and anionic solute species in the rhizosphere of terrestrial plants and highlight the possibility of combining this method with complementary solute imaging techniques.

This protocol presents a workflow for sub-mm 2D visualization of multiple labile inorganic nutrient and contaminant solute species using diffusive gradients in thin films (DGT) combined with mass spectrometry imaging. Solute sampling and high-resolution chemical analysis are described in detail for quantitative mapping of solutes in the rhizosphere of terrestrial plants.