Name: in vitro ovule cultivation for live-cell imaging of zygote polarization and embryo patterning in arabidopsis thaliana
Uploaded: 2017-09-11T14:00:00
Description: Watch this Scientific Journal Video about In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana at JoVE.com

Microscopy in this work was conducted at the Institute of Transformative Bio-Molecules (WPI-ITbM) of Nagoya University and supported by the Japan Advanced Plant Science Network. This work was supported by grants from the Japan Science and Technology Agency (ERATO project to T.H. and M.U.) and from the Japan Society for the Promotion of Science: a Grant-in-Aid for Scientific Research on Innovative Areas (Nos. JP24113514, JP26113710, JP15H05962, and JP15H05955 for M.U., and Nos. JP16H06465, JP16H06464 and JP16K21727 for T.H), a Grant-in-Aid for Young Scientists (B, Nos. JP24770045 and JP26840093 for M.U.), and a Grant-in-Aid for challenging Exploratory Research (No. JP16K14753 for M.U.).

1. Preparation of the In Vitro Ovule Culture Medium
<ol>
	<li>Make the liquid medium forthe in vitro ovule culture (&#34;N5T medium&#34;) containing 1x Nitsch basal salt mixture, 5% (w/v) trehalose dihydrate, 0.05% (w/v) 2-(N-morpholino)ethanesulfonic acid (MES)-KOH (pH 5.8), and 1x Gamborg&#39;s vitamin solution.</li>
	<li>Adjust the pH to 5.8 with KOH.</li>
	<li>Sterilize the medium by autoclaving it (121 &#176;C, 20 min) or filtering it through a 0.22-&#181;m filter. 
	NOTE: The sterilized med...

<ol>
	<li>Natesh, S., Rau, M. A., Johri, 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=.">.</a> Embryology of Angiosperms. , 377-443 (1984).</li><li>Kranz, E., Lorz, H. <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+fertilisation+of+maize+by+single+egg+and+sperm+cell+protoplast+fusion+mediated+by+high+calcium+and+high+pH.">In vitro fertilisation of maize by single egg and sperm cell protoplast fusion mediated by high calcium and high pH.</a> Zygote. 2 (2), 125-128 (1994).</li><li>Uchiumi, T., Uemura, I., Okamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+an+in+vitro+fertilization+system+in+rice+(Oryza+sativa+L.).">Establishment of an in vitro fertilization system in rice (Oryza sativa L.).</a> Planta. 226 (3), 581-589 (2007).</li><li>Sun, M. X., Moscatelli, A., Yang, H. Y., Cresti, M. <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+double+fertilization+in+Nicotiana+tabacum+(L.):+the+role+of+cell+volume+in+cell+fusion.">In vitro double fertilization in Nicotiana tabacum (L.): the role of cell volume in cell fusion.</a> Sex Plant Rep. 13 (4), 225-229 (2001).</li><li>Tian, H. Q., Yuan, T., Russell, 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=Relationship+between+double+fertilization+and+the+cell+cycle+in+male+and+female+gametes+of+tobacco.">Relationship between double fertilization and the cell cycle in male and female gametes of tobacco.</a> Sex Plant Rep. 17 (5), 243-252 (2004).</li><li>Costa, L. M., 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=Central+cell-derived+peptides+regulate+early+embryo+patterning+in+flowering+plants.">Central cell-derived peptides regulate early embryo patterning in flowering plants.</a> Science. 344 (6180), 168-172 (2014).</li><li>Sauer, M., Friml, J. <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+culture+of+Arabidopsis+embryos+within+their+ovules.">In vitro culture of Arabidopsis embryos within their ovules.</a> Plant J. 40 (5), 835-843 (2004).</li><li>Gooh, 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=Live-cell+imaging+and+optical+manipulation+of+Arabidopsis+early+embryogenesis.">Live-cell imaging and optical manipulation of Arabidopsis early embryogenesis.</a> Dev Cell. 34 (2), 242-251 (2015).</li><li>Park, J., Kurihara, D., Higashiyama, T., Arata, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+microcage+arrays+to+fix+plant+ovules+for+long-term+live+imaging+and+observation.">Fabrication of microcage arrays to fix plant ovules for long-term live imaging and observation.</a> Sens Act B-Chem. 191, 178-185 (2014).</li><li>Kimata, Y., 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=Cytoskeleton+dynamics+control+the+first+asymmetric+cell+division+in+Arabidopsis+zygote.">Cytoskeleton dynamics control the first asymmetric cell division in Arabidopsis zygote.</a> Proc Natl Acad Sci U S A. 113 (49), 14157-14162 (2016).</li><li>Nambo, M., 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=Combination+of+Synthetic+Chemistry+and+Live-Cell+Imaging+Identified+a+Rapid+Cell+Division+Inhibitor+in+Tobacco+and+Arabidopsis+thaliana.">Combination of Synthetic Chemistry and Live-Cell Imaging Identified a Rapid Cell Division Inhibitor in Tobacco and Arabidopsis thaliana.</a> Plant Cell Physiol. 57 (11), 2255-2268 (2016).</li><li>Mizuta, Y., Kurihara, D., Higashiyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+with+longer+wavelength+excitation+in+intact+Arabidopsis+tissues.">Two-photon imaging with longer wavelength excitation in intact Arabidopsis tissues.</a> Protoplasma. 252 (5), 1231-1240 (2015).</li></ol>

This manuscript introduces a simple in vitro ovule cultivation protocol that is efficient for use in the live-cell imaging of developing zygotes and embryos.
The design of the PDMS device may need optimization according to the embryo stage. The first developed device was a microcage array to adjust the orientation and to fix the position of the ovules9, and then a micropillar device was constructed to trap ovules more efficiently8. Furth...

The basic body plan of an organism develops from a unicellular zygote. In most flowering plants, zygotic division generates an apical and a basal cell, which develop into the shoot and root, respectively1. Therefore, it is important to understand how the plant body is formed during embryogenesis, but there has not been an effective tool to directly observe the dynamics of living zygotes and embryos because they develop deep in the flower. In several monocot species, such as maize and rice, an in vitro fertilization method has been established2,3. In this method, isolated sperm and egg cells are fused electrically or chemically, and the generated cell can develop into a fertile plant. However, in dicot plants, there is no in vitro fertilization method that can produce proper embryos, presumably because of the non-synchronized cell cycle state of male and female gametes4,5. In addition, the embryo-surrounding tissue (endosperm) plays important roles in embryo development6.
In a model dicot species, A. thaliana, an in vitro cultivation method was developed by focusing on the whole ovule, which contains both the embryo and endosperm7. This system was successfully used to analyze the effects of various chemical reagents on embryogenesis, but it is not suitable for time-lapse imaging because it has a low survival rate. Therefore, a novel in vitro ovule cultivation system was developed in order to start as early as the zygote stage and produce fertile plants at a high ratio8. After various trials, it was found that Nitsch medium and trehalose significantly improved the survival rate of ovules8. In addition, because the ovule expands as it grows and thus frequently moves away from the observation field of the microscope, a PDMS device was developed to fix the ovule in the medium9. The PDMS device enabled the long-term imaging for 3 - 4 days, which is sufficient to trace the development from a zygote to a heart-stage embryo. Using this method, it becomes possible to visualize the dynamics of zygote polarization and embryo patterning, not only under normal conditions, but also in the presence of chemical inhibitors or in various mutant backgrounds8,10,11.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55975/55975fig1.jpg" /> 
Figure 1: Schematic Diagram of the Specific Fluorescent Markers used to Visualize Zygotes and Embryos Through the Ovule. 
The Arabidopsis zygote develops into an embryo in the ovule, which is generated deep inside the flower. In this in vitro cultivation system, the zygote and embryo are observed through the ovule, and thus it is important to use specific fluorescent markers that are not expressed in other ovule tissues. <a href="//cloudflare.jove.com/files/ftp_upload/55975/55975fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table width="606">
	<tbody>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">Nitsch basal salt mixture</td>
			<td style="width:104px;">Duchefa</td>
			<td style="width:119px;">N0223</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">trehalose dihydrate</td>
			<td style="width:104px;">Wako Pure Chemical</td>
			<td style="width:119px;">206-18455</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">MES</td>
			<td style="width:104px;">Dojindo</td>
			<td style="width:119px;">345-01625</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">Gamborg&#8217;s vitamin solution</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">G1019</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">35-mm glass-bottom dish</td>
			<td style="width:104px;">Matsunami Glass</td>
			<td style="width:119px;">D111300</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">35 mm culture dish</td>
			<td style="width:104px;">Corning</td>
			<td style="width:119px;">430588</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">PDMS</td>
			<td style="width:104px;">Dow Corning Co.</td>
			<td style="width:119px;">Sylgard184</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">76 &#215; 26 mm slide glass</td>
			<td style="width:104px;">Matsunami Glass</td>
			<td style="width:119px;">S1225</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">18 &#215; 18 mm slide glass</td>
			<td style="width:104px;">Matsunami Glass</td>
			<td style="width:119px;">C018181</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">needle (gauge 0.40mm)</td>
			<td style="width:104px;">Terumo</td>
			<td style="width:119px;">NN-2719S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">Immersion medium Immersol W 2010</td>
			<td style="width:104px;">Zeiss</td>
			<td style="width:119px;">444969-0000-000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">A1R MP</td>
			<td style="width:104px;">Nikon</td>
			<td style="width:119px;">A1RsiMP(1080) Ti-E-TIRF</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="width:216px;height:84px;">CSU-W1</td>
			<td style="width:104px;">Yokogawa Electric</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">It is a customized equipment, and thus Catalog Number is not avairable.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">CV1000</td>
			<td style="width:104px;">Yokogawa Electric</td>
			<td style="width:119px;">CV1000-SP84</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

in vitro ovule cultivation for live-cell imaging of zygote polarization and embryo patterning in arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

By using this ovule cultivation system, this method can trace the living dynamics of zygote polarization and embryo patterning. This is an achievement because previously there was no technique to visualize the real-time behavior of the zygote and embryo, which are hidden deep in the mother tissue. Figure 3A and Supplemental Video 1 show that the microtubules (MTs) in the young zygote accumulate as a transverse ring in the subapical region<sup...

Watch this Scientific Journal Video about In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana at JoVE.com

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

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