Name: Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species (Video) | JoVE Journal
Uploaded: 2019-03-29T15:00:00.000+00:00
Duration: 6 min 46 s
Description: Learn confocal live imaging of shoot apical meristems in Arabidopsis, tomato, and soybean without fixation or sectioning. This efficient protocol enables high-resolution visualization of cellular structures for plant developmental biology research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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 MS plates and place plates under 4 &#176;C for two days. Then, move MS plates to a short day (8 h light/ 16 h dark), at 22 &#176;C for two weeks.</li>
		<li>Transplant seedlings to soil and grow them in a short day (8 h light/ 16 h dark) at 22 &#176;C for four weeks.</li>
		<li>Transfer plants to continuous light, at 22 &#176;C to induce the transition from the vegetative to the reproductive stage and for imaging the inflorescence SAMs.</li>
	</ol>
	</li>
	<li>Tomato and soybean growth
	<ol>
		<li>Incubate the seeds covered with wet filter paper under 28 &#176;C till they germinate.</li>
		<li>Transplant seedlings to soil and grow them in a long day (16 h light/ 8 h dark) at 25 &#176;C for one week or longer for imaging the vegetative SAMs.</li>
	</ol>
	</li>
</ol>
3. Dissection of the shoot apex
<ol>
	<li>Dissection of the inflorescence shoot apex
	<ol>
		<li>Cut the inflorescence shoot apex together with 1-2 cm main stem from the bolted Arabidopsis plants with a razor blade. Hold the basal part of the main stem and remove as many older flower organs as possible from the main stem by dissecting out the peduncles with jewelry forceps. 
		CAUTION: Avoid cutting fingers when using a razor blade. Dispose of the used razor blades to a proper sharps&#8217; container.</li>
		<li>Hold the attached stem of the shoot apex with jewelry&#160;forceps or fingers in the field of the stereomicroscope, continue removing the rest of flowers till nearly the whole SAM can be viewed from the eyepieces. Remove peduncles clearly at the junction of the main stem.</li>
	</ol>
	</li>
	<li>Dissection of the vegetative shoot apex
	<ol>
		<li>To view the vegetative SAMs from either tomato or soybean, dissect out the cotyledons, leaves, and roots from the plants.</li>
		<li>Hold the hypocotyls of the plants under the stereomicroscope and further dissect out the leaf primordia covering the vegetative SAMs using jewelry forceps.</li>
	</ol>
	</li>
</ol>
4. Staining
<ol>
	<li>To directly visualize cells in SAMs, use freshly prepared propidium iodide (PI) solution to stain cell walls. Dissolve PI powder in sterile, deionized water, make 1mL PI staining solution at the concentration of 1 mg/mL and store the PI solution in microcentrifuge tube covered with aluminum foil.</li>
	<li>Pipette 50 &#181;L PI solution in a clean and empty Petri dish and dip the whole dissected shoot apex into dye for 2 min. Rinse the stained shoot apex twice in sterile, deionized water. During the staining process, immerse the whole inflorescence SAM or vegetative SAM into the PI solution to achieve the uniform staining.</li>
</ol>
5. Image collection
NOTE: For this method, all the SAMs are imaged using an upright confocal microscope and with a 20x water-dipping lens. As described in other protocols9,12,13,15, it is also feasible to image SAMs using an inverted microscope. In addition, the live imaging can be achieved using different brands of confocal microscopes, with the same sample preparation steps. In this study, the imaging steps are described in detail as an example.
<ol>
	<li>Pierce a hole at the center of an imaging dish using forceps and stick the stained shoot apex upright in the medium.</li>
	<li>Fill the imaging dish with sterile, deionized water to completely immerse the sample. Viewing from 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 SAM is fully visible from directly above.</li>
	<li>Place the imaging dish on the sample stage of the confocal microscope. Lower water-dipping lens and raise the microscope sample stage to let the tip of lens dip into the water.</li>
	<li>Open the confocal microscope software and locate the SAM in the brightfield in the eyepieces. Move the SAM sample right below the objective lens through adjusting the XY controller, then focus on the SAM from eyepieces through cautiously adjusting the Z controller.</li>
	<li>Operate the acquisition function in the confocal microscope software (see Table of Materials), start the Live mode to view the sample from the computer screen, and set up all parameters for the laser scanning experiment.
	<ol>
		<li>When adjusting the parameters, use Range Indicator function to define whether the signal is saturated or not.</li>
		<li>Optionally, apply the Reuse function to reload all the parameter settings from the selected confocal file. 
		NOTE: Suggested imaging parameters: Laser line (excitation): 515 nm or 561 nm; Emission 570-650 nm; pinhole: 1 airy unit (AU); Gain: 600-750, scan mode: Frame; Frame size: either 512 X 512 or 1024 X 1024; scanning speed: from 7 to Max; Scanning direction: bi-direction; Averaging number: 2-4; Averaging method: mean; Bit depth: 16 Bit; Scanning Interval: 0.5-1 &#181;M. Further, optimize all these parameters based on the nature of different plant samples and the specific imaging needs.</li>
	</ol>
	</li>
</ol>
6. Image processing
<ol>
	<li>For visualizing optical orthogonal and transverse section views, use the same commercial software for the imaging acquisition. Open the original confocal file, click ortho menu, select ortho. Then select either x position, y position and z position of the image, and save the images as the tiff files.
	<ol>
		<li>Optionally select 3D distance function to define the physical distance between two points that have been selected from the stack of the confocal images.</li>
		<li>Alternatively, use Fiji/Image J, the open resource image processing package to visualize the orthogonal and transverse section views.</li>
		<li>Open the original confocal file with Fiji, click image menu, select Stacks and then select Orthogonal views.</li>
		<li>Select XY, YZ, and XZ planes in the middle position and save as Tiff format images.</li>
	</ol>
	</li>
	<li>For visualizing a 3D transparent projection, use the same software.
	<ol>
		<li>Open the original confocal file, click 3D menu, and select Transparent to generate a 3D projection view.</li>
		<li>Optionally click 3D menu, select Appearance, and then select Transparency to adjust three parameters of the projection including Threshold, Ramp and Maximum for the transparency of the 3D image.</li>
		<li>Click 3D menu, select Appearance, and select Light to adjust the brightness of the 3D image.</li>
		<li>Export the projected images and save them as the Tiff files.</li>
	</ol>
	</li>
	<li>For visualizing a 3D maximum intensity projection, open the confocal files with the same software, and click the 3D menu.
	<ol>
		<li>Select 3D menu and select Maximum.</li>
		<li>Alternatively, use Fiji/Image J to visualize a 3D maximum intensity projection.</li>
		<li>Open the original confocal file with Fiji, click Image menu and select stacks.</li>
		<li>Select 3D project and save as Tiff format images.</li>
	</ol>
	</li>
	<li>For visualizing the depth coding view of the 3D images, use the same software.
	<ol>
		<li>Click 3D menu and select Appearance.</li>
		<li>Select Special and select Depth Coding.</li>
		<li>Alternatively, use Fiji/Image J to visualize the depth coding view.</li>
		<li>Open the confocal files, click image menu, select Hyperstack.</li>
		<li>Select Temporal-Color code and save as Tiff format images. 
		NOTE: The depth coding z-stack also can be achieved through the plugin Z Code Stack for Fiji.</li>
	</ol>
	</li>
	<li>For visualizing the 3D rotation view as shown (Movie 1 and Movie 2), use the same software (See the Table of Materials).
	<ol>
		<li>Open the confocal files, click 3D menu, and select Series.</li>
		<li>Select Render series, and select one of the four options including Turn around x, Turn around y, Start and end, and Position list.</li>
		<li>Save the render series as the AVI files.</li>
		<li>Alternatively, use Fiji/Image J to visualize the 3D rotation view.</li>
		<li>Open the original confocal file with Fiji, click Image menu, and select Stacks.</li>
		<li>Select 3D project and save as AVI format videos.</li>
	</ol>
	</li>
</ol>

<ol><li>Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+control+of+cell+division+patterns+in+developing+plants%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Genetic control of cell division patterns in developing plants.</a> Cell. 88 (3), 299-308 (1997).</li>
<li>Xu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+cascade+of+arabinosyltransferases+controls+shoot+meristem+size+in+tomato%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">A cascade of arabinosyltransferases controls shoot meristem size in tomato.</a> Nature Genetics. 47 (7), 784-792 (2015).</li>
<li>Bommert, P., Nagasawa, N. S., Jackson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+variation+in+maize+kernel+row+number+is+controlled+by+the+FASCIATED+EAR2+locus%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">Quantitative variation in maize kernel row number is controlled by the FASCIATED EAR2 locus.</a> Nature Genetics. 45 (3), 334-337 (2013).</li>
<li>Je, B. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Signaling+from+maize+organ+primordia+via+FASCIATED+EAR3+regulates+stem+cell+proliferation+and+yield+traits%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">Signaling from maize organ primordia via FASCIATED EAR3 regulates stem cell proliferation and yield traits.</a> Nature Genetics. 48 (7), 785-791 (2016).</li>
<li>Ping, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dt2+is+a+gain-of-function+MADS-domain+factor+gene+that+specifies+semideterminacy+in+soybean%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">Dt2 is a gain-of-function MADS-domain factor gene that specifies semideterminacy in soybean.</a> Plant Cell. 26 (7), 2831-2842 (2014).</li>
<li>Vaughan, J. G., Jones, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+of+the+angiosperm+inflorescence+apex%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Structure of the angiosperm inflorescence apex.</a> Nature. 171, 751(1953).</li>
<li>Sijacic, P., Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+insights+from+live-imaging+in+shoot+meristem+development%5BTitle%5D)+AND+%22Journal+of+Integrative+Plant+Biology%22%5BJournal%5D)">Novel insights from live-imaging in shoot meristem development.</a> Journal of Integrative Plant Biology. 52 (4), 393-399 (2010).</li>
<li>Tax, F. E., Durbak, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Meristems+in+the+movies%3A+live+imaging+as+a+tool+for+decoding+intercellular+signaling+in+shoot+apical+meristems%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">Meristems in the movies: live imaging as a tool for decoding intercellular signaling in shoot apical meristems.</a> Plant Cell. 18 (6), 1331(2006).</li>
<li>Grandjean, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+analysis+of+cell+division%2C+cell+growth%2C+and+differentiation+at+the+shoot+apical+meristem+in+Arabidopsis%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">In vivo analysis of cell division, cell growth, and differentiation at the shoot apical meristem in Arabidopsis.</a> Plant Cell. 16 (1), 74-87 (2004).</li>
<li>Heisler, M. G., Ohno, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Live-imaging+of+the+Arabidopsis+inflorescence+meristem%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Live-imaging of the Arabidopsis inflorescence meristem.</a> Methods in Molecular Biology. 1110, 431-440 (2014).</li>
<li>Tobin, C. J., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-time+lineage+analysis+to+study+cell+division+orientation+in+the+Arabidopsis+shoot+meristem%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Real-time lineage analysis to study cell division orientation in the Arabidopsis shoot meristem.</a> Methods in Molecular Biology. 1370, 147-167 (2016).</li>
<li>Prunet, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Live+confocal+Imaging+of+developing+Arabidopsis+flowers%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Live confocal Imaging of developing Arabidopsis flowers.</a> Journal of Visualized Experiments. (122), e55156(2017).</li>
<li>Prunet, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Live+confocal+imaging+of+Arabidopsis+flower+buds%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Live confocal imaging of Arabidopsis flower buds.</a> Developmental Biology. 419, 114-120 (2016).</li>
<li>Reddy, G. V., Heisler, M. G., Ehrhardt, D. W., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-time+lineage+analysis+reveals+oriented+cell+divisions+associated+with+morphogenesis+at+the+shoot+apex+of+Arabidopsis+thaliana%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Real-time lineage analysis reveals oriented cell divisions associated with morphogenesis at the shoot apex of Arabidopsis thaliana.</a> Development. 131, 4225-4237 (2004).</li>
<li>Nimchuk, Z. L., Perdue, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Live+Imaging+of+Shoot+Meristems+on+an+Inverted+Confocal+Microscope+Using+an+Objective+Lens+Inverter+Attachment%5BTitle%5D)+AND+%22Frontiers+in+Plant+Science%22%5BJournal%5D)">Live Imaging of Shoot Meristems on an Inverted Confocal Microscope Using an Objective Lens Inverter Attachment.</a> Frontiers in Plant Science. 8, 773(2017).</li>
<li>Zhou, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HAIRY+MERISTEM+with+WUSCHEL+confines+CLAVATA3+expression+to+the+outer+apical+meristem+layers%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">HAIRY MERISTEM with WUSCHEL confines CLAVATA3 expression to the outer apical meristem layers.</a> Science. 361 (6401), 502-506 (2018).</li>
<li>Zhou, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Control+of+plant+stem+cell+function+by+conserved+interacting+transcriptional+regulators%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Control of plant stem cell function by conserved interacting transcriptional regulators.</a> Nature. 517 (7534), 377-380 (2015).</li>
<li>Nimchuk, Z. L., Zhou, Y., Tarr, P. T., Peterson, B. A., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plant+stem+cell+maintenance+by+transcriptional+cross-regulation+of+related+receptor+kinases%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Plant stem cell maintenance by transcriptional cross-regulation of related receptor kinases.</a> Development. 142 (6), 1043(2015).</li>
<li>Li, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((LEAFY+Controls+Auxin+Response+Pathways+in+Floral+Primordium+Formation%5BTitle%5D)+AND+%22Science+Signaling%22%5BJournal%5D)">LEAFY Controls Auxin Response Pathways in Floral Primordium Formation.</a> Science Signaling. 6 (270), ra23(2013).</li>
<li>Truernit, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-resolution+whole-mount+imaging+of+three-dimensional+tissue+organization+and+gene+expression+enables+the+study+of+phloem+development+and+structure+in+Arabidopsis%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">High-resolution whole-mount imaging of three-dimensional tissue organization and gene expression enables the study of phloem development and structure in Arabidopsis.</a> Plant Cell. 20 (6), 1494-1503 (2008).</li>
</ol>

The authors have nothing to disclose.

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

confocal live imaging of shoot apical meristems from different plant species

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.
<p class="jove_content" fo:keep-together.withi...

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 ...

confocal-live-imaging-shoot-apical-meristems-from-different-plant

Research

JoVE Journal

Developmental Biology

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.<br...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

Live Confocal Imaging of Developing Arabidopsis Flowers

Presented here is a simple technique for high-resolution confocal time-lapse imaging of root and hypocotyl development for up to 3 days using high numerical-aperture objectives and perfluorodecalin as an immersion medium.

A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here we describe a protocol for live cell imaging of the cortical microtubule cytoskeleton at the shoot apical meristem and monitoring its response to changes in physical forces.

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

This manuscript presents a detailed protocol to image the 3-D cell wall dynamics of living moss tissue, allowing the visualization of the detachment of cell walls in ggb mutants and thickening cell wall patterns in the wild type during development over a long period.

3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss Physcomitrium patens

Maize leaf primordia are deeply ensheathed and rolled, making them difficult to study. Here, we present methods for preparing transverse sections and unrolled whole mounts of maize leaf primordia for fluorescence and confocal imaging.

Improved Methods for Preparing Transverse Sections and Unrolled Whole Mounts of Maize Leaf Primordia for Fluorescence and Confocal Imaging

Chromosome movement during meiotic prophase is a poorly understood phenomenon that is fundamental to spore formation. This protocol describes a method to acquire, analyze, and quantify centromere movements during meiotic prophase in Arabidopsis thaliana male meiocytes.

Live Imaging Characterization of Centromere Movements During Male Meiotic Prophase in Arabidopsis thaliana

This process uses confocal microscopy in conjunction with a fluorescently labeled nucleus and chromatin marker line in Arabidopsis thaliana to simultaneously record nucleus and chromatin dynamics in Arabidopsis roots and investigate changes in these dynamics in response to developmental and environmental cues.

Quantitative Live Cell Imaging of Nuclear Shape and Chromatin Dynamics During Development and Environmental Stress in Arabidopsis thaliana Root

Learn confocal live imaging of shoot apical meristems in Arabidopsis, tomato, and soybean without fixation or sectioning. This efficient protocol enables high-resolution visualization of cellular structures for plant developmental biology research.

Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species

Explore More Videos