Name: confocal live imaging of shoot apical meristems from different plant species
Uploaded: 2019-03-29T15:00:00
Description: Watch this Scientific Journal Video about Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species at JoVE.com

The authors acknowledge Purdue Bindley Bioscience Center Imaging Facility for accessing the laser scanning confocal microscope and for the technical support, and the authors appreciate the help from Andy Schaber in the Purdue Bindley Imaging Facility. This activity was funded by Purdue University as part of AgSEED Crossroads funding to support Indiana&#8217;s Agriculture and Rural Development.

1. Media and imaging dishes preparation
<ol>
	<li>MS plates: Add 0.5x Murashige &#38; Skoog MS medium, 1% agar into deionized water and then adjust pH to 5.8 using potassium hydroxide solution (Optional: Add 1% sucrose for the long-term plant growing). Autoclave and pour plates.</li>
	<li>Imaging dishes: Fill plastic Petri dishes (6 cm wide, 1.5 cm depth) to 0.5-0.8 cm with 1.5% molten agarose.</li>
</ol>
2. Plant growth
<ol>
	<li>Arabidopsis growth
	<ol>
		<li>Sow sterilized seeds on...

<ol>
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Here, we describe a simple imaging method that can be applied to the study of shoot apical meristems from different plants with minor modification, opening a new avenue to study the meristem regulation at both vegetative and reproductive stages in model plants and crops. In contrast to the SEM and histological staining methods, this protocol can help reveal both surface view and internal cellular structures of the SAMs, without the need for labor-intensive sample fixation and/or tissue sectioning steps. This protocol is ...

The plant meristem contains a pool of undifferentiated stem cells and continuously sustains the plant organ growth and development1. During the postembryonic development, almost all aboveground tissues of a plant are derived from the shoot apical meristem (SAM). In crops, the activity and size of the SAM and its derived floral meristems are tightly associated with many agronomic traits such as shoot architecture, fruit production, and seed yield. For example, in tomato, an enlarged SAM causes an increase in the shoot and inflorescence branching, and thus results in generating extra flower and fruit organs2. In maize, an increase in SAM size leads to a higher seed number and total yield3,4. In soybean, the meristem indeterminacy is also closely associated with the shoot architecture and yield5.
The morphology and anatomy of SAMs can be characterized by several different methods, including histological sectioning/staining and scanning electron microscopy (SEM)6, both of which have greatly advanced the meristem research through providing either the sectional view or a three-dimensional (3D) surface view of SAMs. However, both methods are time consuming, involving several experimental steps from the sample preparation to the data acquisition, and these methods mainly depend on fixed samples. Recent advances in laser scanning confocal microscopy technique have overcome these limitations and provide us with a powerful tool to investigate the cellular structure and developmental process of plant tissues and organs7,8. Through optical rather than physical tissue sectioning, confocal microscopy allows the collection of a series of z-stack images and the subsequent 3D reconstruction of the sample through image analysis software.
Here, we describe an efficient procedure for investigating both inside and surface structures of the living SAMs from different plant species using laser scanning confocal microscopy, which potentially allows researchers to accomplish all the experimental process within as short as 20 minutes. Different from other published methods for live confocal imaging of Arabidopsis inflorescence SAMs9,10,11,12,13,14,15 and Arabidopsis flowers12,13, here we demonstrate that this protocol is highly efficient for studying not only the inflorescence meristems of the model species but also the vegetative shoot apical meristems from different crops, such as tomato and soybean. This method does not rely on transgenic fluorescent markers, and potentially can be applied to study the shoot meristems from many different species and cultivars. In addition, we also introduce the simple image processing steps for viewing and analyzing different SAMs in a&#160;3D view. Taken together, this simple method will facilitate researchers better understanding both the structure and developmental process of the meristems from both model organisms and crops.

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	<tbody>
		<tr height="32">
			<td height="32" style="height:32px;width:283px;">Agar Phyto</td>
			<td style="width:176px;">Dot Scientific Inc.</td>
			<td style="width:119px;">DSA20300-1000</td>
			<td style="width:391px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:35px;width:283px;">Agarose</td>
			<td style="width:176px;">Dot Scientific Inc.</td>
			<td style="width:119px;">AGLE-500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:283px;">Forceps&#160;</td>
			<td style="width:176px;">ROBOZ</td>
			<td style="width:119px;">RS-4955</td>
			<td>Dumont #5SF Super Fine Forceps Inox Tip Size .025 X .005mm, for dissecting shoot apices.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:283px;">LSM 880 Upright Confocal Microscope&#160;</td>
			<td style="width:176px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Murashige &#38; Skoog MS medium</td>
			<td style="width:176px;">Dot Scientific Inc.</td>
			<td style="width:119px;">DSM10200-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Plan APO 20x/1.1 water dipping lens</td>
			<td style="width:176px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:283px;">Plastic petri dishes 100 mm X 15 mm</td>
			<td style="width:176px;">CELLTREAT Scientific Products</td>
			<td style="width:119px;">229694</td>
			<td style="width:391px;">Use as making MS plates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Plastic petri dishes60 mm X 15</td>
			<td style="width:176px;">CELLTREAT Scientific Products</td>
			<td style="width:119px;">229665</td>
			<td>Use as imaging dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Propagation Mix</td>
			<td style="width:176px;">Sungro Horticulture</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Propidium iodide</td>
			<td style="width:176px;">Acros Organics</td>
			<td style="width:119px;">440300250</td>
			<td>1 mg/mL solution in water, to stain the cell walls</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Razor blade</td>
			<td style="width:176px;">PERSONNA&#160;</td>
			<td style="width:119px;">62-0179</td>
			<td style="width:391px;">For cutting shoot apex from plants</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:35px;width:283px;">Stereomicroscope</td>
			<td style="width:176px;">Nikon</td>
			<td style="width:119px;">SMZ1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Tissue</td>
			<td style="width:176px;">VWR</td>
			<td style="width:119px;">82003-820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Zen black</td>
			<td style="width:176px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td>Image acquisition software</td>
		</tr>
	</tbody>
</table>

confocal live imaging of shoot apical meristems from different plant species

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic development. The activity and morphology of SAMs determine important agronomic traits, such as shoot architecture, size and number of reproductive organs, and most importantly, grain yield. Here, we provide a detailed protocol for analyzing both the surface morphology and the internal cellular structure of the living SAMs from different species through laser scanning confocal microscope. The whole procedure from the sample preparation to the acquisition of high resolution three-dimensional (3D) images can be accomplished within as short as 20 minutes. We demonstrate that this protocol is highly efficient for studying not only the inflorescence SAMs of the model species but also the vegetative meristems from different crops, providing a simple but powerful tool to study the organization and development of meristems across different plant species.

To evaluate the efficiency of our protocol and to explore the morphology of the meristems from different species, we have performed the confocal live imaging experiments on the inflorescence meristem from Arabidopsis and the vegetative meristems from both tomato and soybean. In this study, Arabidopsis ecotype Landsberg erecta, tomato cultivar Micro-Tom and soybean cultivar Williams 82 have been used as examples.
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Watch this Scientific Journal Video about Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species at JoVE.com

Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species

This protocol presents how to live image and analyze the shoot apical meristems from different plant species using laser scanning confocal microscopy.

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic ...

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