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>
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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.

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8.9K Views.  Toyo University. 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 be...