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...

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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.

<table border="0" cellpadding="0" cellspacing="0" style="width:968px;" width="968">
	<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 ...

Shoot apical meristems control plant growth and reproduction. This method helps us investigate the morphology and internal structures of shoot apical meristems not only in the model species but also in different crops. This technique is a very efficient procedure.Shoot apical meristem can be directly visualized in a high cellular resolution without any labor-intensive sample fixation and tissue sectioning steps. To begin, grow Arabidopsis, tomato, and soybean plants as detailed in the text protocol. Cut the inflorescence shoot apex from the bolted Arabidopsis plants.Hold the basal part of the main stem, and use forceps to remove as many older flower organs as possible from the main stem. Continue removing the rest of the flowers in the field of the stereo microscope until the shoot apical meristem can be viewed from the eyepieces. To view the vegetative shoot apical meristems from either tomato or soybean, dissect out the cotyledons, leaves, and roots from the plant.Hold the hypocotyls of the plant under the stereo microscope, and use forceps to further dissect out the leaves and leaf primordia covering the vegetative shoot apical meristems. Pipette 50 microliters of propidium iodide solution in a clean and empty Petri dish. Dip the whole dissected shoot apex into dye for two minutes.During the staining process, immerse the whole inflorescence shoot apical meristem or vegetative shoot apical meristem into the propidium iodide solution to achieve uniform staining. Rinse the stained shoot apex twice in sterile, deionized water. Now, pierce a hole at the center of a prepared imaging dish using forceps, and stick the stained shoot apex upright in the medium.Fill the imaging dish with sterile, deionized water to completely immerse the sample. View the sample through the stereo microscope. Pipette up and down to remove air bubbles trapped around the meristem.Then, adjust the angle of the stem in the agar to make sure that the shoot apical meristem is fully visible from directly above. Now, place the imaging dish on the sample stage of the confocal microscope. Lower the water-dipping lens, and raise the microscope sample stage to let the tip of lens dip into the water.Open the confocal microscope software, and use the eyepieces to locate and focus on the shoot apical meristem in the bright field. Operate the Acquisition function in the confocal microscope software. Start the live mode to view the sample from the computer screen, and set up all parameters for the laser scanning experiment.When adjusting the parameters, use the Range Indicator function to define whether the signal is saturated or not. The same software that was used for imaging acquisition can be used for visualizing a three-dimensional transparent projection. Open the original confocal file, click 3D menu, and select Transparent to generate a 3D projection view.Optionally, select Transparency to adjust three parameters of the projection, including Threshold, Ramp, and Maximum for the transparency of the 3D image. Now, select Light to adjust the brightness of the 3D image. Export the projected images, and save them as TIFF files.For visualizing the depth coding view of the 3D images, use the same software. Again, click 3D menu, and select Appearance. Select Special, and select Depth Coding.Export the projected images, and save them as TIFF files. This image from the orthogonal section through the middle of the Arabidopsis shoot apical meristem shows that the horizontal walls are stained with propidium iodide in almost all the cells at multiple cell layers. A view of one transverse section through the corpus of the inflorescence shoot apical meristem shows that the cells from the XY plane are also clearly imaged.The 3D projection view reveals that the inflorescence meristem forms a dome-like structure and is surrounded by the developing flower primordia. This image from the orthogonal section through the middle of the tomato shoot apical meristem also shows that the horizontal walls are stained with propidium iodide at multiple cell layers, although the propidium iodide signal from the deep interior region is slightly lower. An image from one transverse section through the deep layers of the vegetative shoot apical meristem reveals the cells from the XY planes and the boundary formed between the vegetative meristem and leaf primordia.The 3D projection view can further provide a comprehensive view of the shape and organization of the vegetative meristem from tomato. Shown here is an orthogonal view through the middle of the soybean shoot apical meristem and a 3D projection of the same shoot apical meristem. The dome-like vegetative meristem and its derived new leaf primordia are also observable.Propidium iodide can easily stain damaged or dead cells, which will greatly influence the quality of images, so it's important to avoid any physical damage while preparing shoot apical meristem samples.

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Developmental Biology

12.0K visualizações.  Purdue University. Atire em meristems apical controle o crescimento e a reprodução das plantas. Este método nos ajuda a investigar a morfologia e as estruturas internas de meristems apical shoot não apenas nas espécies modelo, mas também em diferentes culturas. Esta técnica é um procedimento muito eficiente.O meristem apical de tiro pode ser diretamente visualizado em uma alta resolução celular sem qualquer fixação de amostra intensiva em mão-de-obra e etapas de secção de tecidos. Para com...