Name: micron-scale phenotyping techniques of maize vascular bundles based on x-ray microcomputed tomography
Uploaded: 2018-10-09T15:00:00
Description: Watch this Scientific Journal Video about Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography at JoVE.com

This research was supported by the National Nature Science Foundation of China (No.31671577), the Science and Technology Innovation Special Construction Funded Program of Beijing Academy of Agriculture and Forestry Sciences(KJCX20180423), the Research Development Program of China (2016YFD0300605-01), the Beijing Natural Science Foundation (5174033), the Beijing Postdoctoral Research Foundation (2016 ZZ-66), and the Beijing Academy of Agricultural and Forestry Sciences Grant (KJCX20170404), (JNKYT201604).

1. Sample Preparation Protocol
<ol>
	<li>For the sampling, collect the stem, leaf, and root from fresh maize plants and divide them into three types of sample groups (each group with four replications). Then, cut them into small segments using a surgical blade in the following manner: (1) cut a segment of the middle stem internode 1 - 1.5 cm in length; (2) cut a segment of the maximum width of the leaf 0.5 - 3 cm in length along the vertical direction with the main vein; (3) cut a segment of the crown root 0.5 cm in le...

<ol>
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R., Mertens, D. R., Hatfield, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolates+of+cell+types+from+sorghum+stems:+Digestion,+cell+wall+and+anatomical+characteristics.">Isolates of cell types from sorghum stems: Digestion, cell wall and anatomical characteristics.</a> Journal of the Science of Food and Agriculture. 63, 407-417 (1993).</li><li>Hatfield, R., Wilson, J., Mertens, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition+of+cell+walls+isolated+from+cell+types+of+grain+sorghum+stems.">Composition of cell walls isolated from cell types of grain sorghum stems.</a> Journal of the Science of Food and Agriculture. 79, 891-899 (1999).</li><li>Du, J., 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=Micron-scale+phenotyping+quantification+and+three-dimensional+microstructure+reconstruction+of+vascular+bundles+within+maize+stems+based+on+micro-CT+scanning.">Micron-scale phenotyping quantification and three-dimensional microstructure reconstruction of vascular bundles within maize stems based on micro-CT scanning.</a> Functional Plant Biology. 44 (1), 10-22 (2016).</li><li>Pan, X., 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=Reconstruction+of+Maize+Roots+and+Quantitative+Analysis+of+Metaxylem+Vessels+based+on+X-ray+Micro-Computed+Tomography.">Reconstruction of Maize Roots and Quantitative Analysis of Metaxylem Vessels based on X-ray Micro-Computed Tomography.</a> Canadian Journal of Plant Science. 98 (2), 457-466 (2018).</li><li>McElrone, A. J., Choat, B., Parkinson, D. Y., MacDowell, A. A., Brodersen, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+high+resolution+computed+tomography+to+visualize+the+three+dimensional+structure+and+function+of+plant+vasculature.">Using high resolution computed tomography to visualize the three dimensional structure and function of plant vasculature.</a> Journal of Visualized Experiments. (74), e50162 (2013).</li><li>Cloetens, P., Mache, R., Schlenker, M., Lerbs-Mache, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+phase+tomography+of+Arabidopsis+seeds+reveals+intercellular+void+network.">Quantitative phase tomography of Arabidopsis seeds reveals intercellular void network.</a> Proceedings of the National Academy of Sciences of the Unites States of America. 103, 14626-14630 (2006).</li><li>Dorca-Fornell, C., 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=Increased+leaf+mesophyll+porosity+following+transient+retinoblastoma-related+protein+silencing+is+revealed+by+microcomputed+tomography+imaging+and+leads+to+a+system-level+physiological+response+to+the+altered+cell+division+pattern.">Increased leaf mesophyll porosity following transient retinoblastoma-related protein silencing is revealed by microcomputed tomography imaging and leads to a system-level physiological response to the altered cell division pattern.</a> Plant Journal. 76 (6), 914-929 (2013).</li><li>Verboven, P., 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=Void+space+inside+the+developing+seed+of+Brassica+napus+and+the+modelling+of+its+function.">Void space inside the developing seed of Brassica napus and the modelling of its function.</a> New Phytologist. 199, 936-947 (2013).</li><li>Brodersen, C. R., Roark, L. C., Pittermann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiological+implications+of+primary+xylem+organization+in+two+ferns.">The physiological implications of primary xylem organization in two ferns.</a> Plant, Cell &amp; Environment. 35, 1898-1911 (2012).</li><li>Choat, B., Brodersen, C. R., McElrone, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchrotron+X-ray+microtomography+of+xylem+embolism+in+Sequoia+sempervirens+saplings+during+cycles+of+drought+and+recovery.">Synchrotron X-ray microtomography of xylem embolism in Sequoia sempervirens saplings during cycles of drought and recovery.</a> New Phytologist. 205, 1095-1105 (2015).</li><li>Torres-Ruiz, J. 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=Direct+x-ray+microtomography+observation+confirms+the+induction+of+embolism+upon+xylem+cutting+under+tension.">Direct x-ray microtomography observation confirms the induction of embolism upon xylem cutting under tension.</a> Plant Physiology. 167, 40-43 (2015).</li><li>Staedler, Y. M., Masson, D., Sch&#246;nenberger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+tissues+in+3D+via.+x-ray+tomography:+simple+contrasting+methods+allow+high+resolution+imaging.">Plant tissues in 3D via. x-ray tomography: simple contrasting methods allow high resolution imaging.</a> PLoS One. 8, 75295 (2013).</li><li>Zhang, Y., Legay, S., Barri&#232;re, Y., M&#233;chin, V., Legland, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Color+quantification+of+stained+maize+stem+section+describes+lignin+spatial+distribution+within+the+whole+stem.">Color quantification of stained maize stem section describes lignin spatial distribution within the whole stem.</a> Journal of the Science of Food and Agriculture. 61, 3186-3192 (2013).</li><li>Legland, D., Devaux, M. F., Guillon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+mapping+of+maize+bundle+intensity+at+the+stem+scale+using+spatial+normalisation+of+replicated+images.">Statistical mapping of maize bundle intensity at the stem scale using spatial normalisation of replicated images.</a> PLoS One. 9 (3), 90673 (2014).</li><li>Heckwolf, S., Heckwolf, M., Kaeppler, S. M., de Leon, N., Spalding, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+analysis+of+anatomical+traits+in+stem+transections+of+maize+and+other+grasses.">Image analysis of anatomical traits in stem transections of maize and other grasses.</a> Plant Methods. 11, 26 (2015).</li><li>Wu, H., Jaeger, M., Wang, M., Li, B., Zhang, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+distribution+of+vessels,+passage+cells+and+lateral+roots+along+the+root+axis+of+winter+wheat+(Triticum+aestivum).">Three-dimensional distribution of vessels, passage cells and lateral roots along the root axis of winter wheat (Triticum aestivum).</a> Annals of Botany. 107, 843-853 (2011).</li><li>Chopin, J., Laga, H., Huang, C. Y., Heuer, S., Miklavcic, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RootAnalyzer:+A+Cross-Section+Image+Analysis+Tool+for+Automated+Characterization+of+Root+Cells+and+Tissues.">RootAnalyzer: A Cross-Section Image Analysis Tool for Automated Characterization of Root Cells and Tissues.</a> PLoS One. 10, 0137655 (2015).</li><li>Passot, S., 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=Characterization+of+pearl+millet+root+architecture+and+anatomy+reveals+three+types+of+lateral+roots.">Characterization of pearl millet root architecture and anatomy reveals three types of lateral roots.</a> Frontiers in Plant Science. 7, 829 (2016).</li></ol>

With the successful application of CT technology in the fields of biomedicine and materials science, this technology has been gradually introduced into the fields of botany and agriculture, promoting researches in plant life sciences as a promising technical tool. In the late 1990s, CT technology was first used to study the morphological structures and development of plant root systems. In the past decade, synchrotron HRCT has become a powerful, nondestructive tool for plant biologists, and has been successfully used to ...

The maize vascular system runs through the entire plant, from the root and stem to the leaves, which forms the key transportation paths for delivering water, mineral nutrients, and organic substances1. Another important function of the vascular system is to provide mechanical support for the maize plant. For example, the morphology, number, and distribution of vascular bundles in roots and stems are closely related to the lodging resistance of maize plants2,3. At present, studies on the anatomical structure of vascular bundles mainly utilize microscopic and ultramicroscopic techniques to display the anatomical structures of a certain part of the stem, leaf, or root, and then measure and count these structures of interest by manual investigation. Undoubtedly, manual measurement of various microscopic structures in large-scale microimages is a very tedious and inefficient work and severely limits the precision of microphenotypic traits, owing to its subjectivity and inconsistency4,5.
Maize has no secondary growth, and the cell content essentially consists of water in the primary meristem. Without any pretreatment, fresh samples of maize tissues can be directly scanned using a micro-CT device; however, the scanning results are probably poor and rough. The main reasons are summarized as follows: (1) low attenuation densities of plant tissues, resulting in a low contrast of atomic number and high noise in images; (2) fresh plant materials are prone to dehydrate and shrink during the normal scanning environment, as reported by Du6. The abovementioned problems have become the main constraints for the development and application of microphenotyping technology for maize, wheat, rice, and other monocotyledons. Here, we introduce the &#39;sample preparation protocol&#39; to pretreat the samples of maize stem, leaf, and root. This protocol avoids the dehydration and deformation of plant materials during the CT scanning; thus, it is beneficial to increase the preservation time of plant samples with nondeformation. Moreover, the dyeing step based on solid iodine also enhances the contrast of plant materials; thus, it makes significant improvements in the imaging quality of micro-CT. Furthermore, we developed image processing software, named VesselParser, to process the CT images of maize stems and leaves. This software integrates a set of image-processing pipelines to perform high-throughput and automatic phenotyping analysis for 2-D CT images of different plant tissues. Vascular bundles in the entire cross-section of the maize stem and leaf are detected, extracted, and identified using an automatic image-processing method. As a result, we obtain 31 microscopic phenotypes of the maize stem and 33 microscopic phenotypes of the maize leaf. For the CT image series of the maize root, we developed an image-processing scheme to acquire 3-D phenotypic traits of metaxylem vessels. This scheme is superior in efficiency of image acquisition and reconstruction compared with traditional methods.
These results indicate that the image processing pipelines considering the imaging characteristics of ordinary X-ray micro-CT provide an effective method for the microscopic phenotyping of vascular bundles; this extremely widens the applications of CT techniques in plant science and improves the automatic phenotyping of plant materials at cellular resolution6,7.

<table border="0" cellpadding="0" cellspacing="0" style="width:841px;" width="841">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Skyscan 1172 X-ray computed tomography system</td>
			<td style="width:269px;">Bruker Corporation, Belgium</td>
			<td style="width:119px;">NA</td>
			<td style="width:237px;">For CT scanning</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:216px;">CO2 critical point drying system (Leica CPD300)</td>
			<td style="width:269px;">Leica Corporation, Germany</td>
			<td style="width:119px;">NA</td>
			<td style="width:237px;">For sample drying</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:269px;">Any</td>
			<td style="width:119px;">NA</td>
			<td>For FAA fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Formaldehyde</td>
			<td style="width:269px;">Any</td>
			<td style="width:119px;">NA</td>
			<td>For FAA fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acetic acid</td>
			<td style="width:269px;">Any</td>
			<td style="width:119px;">NA</td>
			<td>For FAA fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical blade</td>
			<td style="width:269px;">Any</td>
			<td style="width:119px;">NA</td>
			<td>For cutting the sample sgements</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">3D printer</td>
			<td style="width:269px;">Makerbot replicator 2, MakerBot Industries, USA</td>
			<td style="width:119px;">NA</td>
			<td>For printing the sample baskets of maize root, stem, and leaf</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge tube</td>
			<td style="width:269px;">Corning, USA</td>
			<td style="width:119px;">NA</td>
			<td>Place the root, stem, or leaf materials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Solid iodine</td>
			<td style="width:269px;">Any</td>
			<td style="width:119px;">NA</td>
			<td>For sample dyeing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SkyScan Nrecon software</td>
			<td style="width:269px;">SkyScan NRecon, Version: 1.6.9.4, Bruker Corporation, Belgium</td>
			<td style="width:119px;">NA</td>
			<td>For image reconstruction</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">VesselParser software</td>
			<td style="width:269px;">VesselParser, Version: 3.0, National Engineering Research Center for Information Technology in Agriculture (NERCITA), Beijing, China</td>
			<td style="width:119px;">NA</td>
			<td>Image analysis protocol for single CT image of maize stem or leaf</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ScanIP</td>
			<td style="width:269px;">ScanIP, Version: 7.0; Simpleware, Exeter, UK</td>
			<td style="width:119px;">NA</td>
			<td>3D image processing software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Latex gloves</td>
			<td style="width:269px;">Any</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tweezers</td>
			<td style="width:269px;">Any</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

micron-scale phenotyping techniques of maize vascular bundles based on x-ray microcomputed tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

The sample preparation protocol suitable for ordinary micro-CT scanning not only prevents the deformation of plant tissues but also enhances the X-ray absorption contrast. Pretreated plant materials are scanned using a micro-CT system into high-quality slice images, and the highest resolution can reach 2 &#181;m/pixel. Figure 4 shows the scanned micro-CT images of stem, leaf, and root, and the image contrast has a significant improvement compared with the res...

Watch this Scientific Journal Video about Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography at JoVE.com

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

This method can have answer quick questions in the planned micron-scale phenotyping techniques build. Such as micro-CT scanning particles, and the phenotypic analysis method for maize vascular bundles. The main advantage of this technique is to provide new and effective ways to investigate phenotypical traits of maize vascular bundle by sample preparation, CT scanning, and analysis of protocols.The implications of this technique broadens applications of ordinary micro-CT scanning and other plant sciences. This protocol can be easily modified to accommodate other plant materials, such as dehydration of drying procedure. To begin, collect the stem, leaf, and root from fresh maize plants.And divide them into three sample groups. Next, use a surgical blade to cut a one to 1.5 centimeter segment of the middle stem internode. Using the same surgical blade, cut a 1/2 to two centimeter segment from the maximum width of the leaf along the vertical direction with the main vein.Use the surgical blade to cut a 1/2 centimeter segment from the ground root. After this, soak the sample segments in FAA solution for at least three days. Dehydrate the sample segments for 30 minutes in six sequential ethanol gradients.Then place the plant materials in the corresponding sample baskets. Quickly transfer the baskets to the sample cell of a carbon dioxide critical point drying system. Place the dried plant materials into a 50 milliliter centrifuge tube with two grams of solid iodine.Then place the tubes in a light-proof room for four to five hours. First, set the CT scanning parameters according to the text protocol. Then, set the scanning ranges according to the different sizes and volumes of the plant materials used.Next, adjust the imaging pixel sizes as follows:Two micrometers for the root. 6.77 micrometers for the stem. And 10 micrometers for the leaf.To reconstruct slice images, use imagery construction software to convert the raw CT data into CT slice images with 2K resolution. First, specify the organ type to initialize different algorithm pipelines in the automatic imaging software. Then, click the method parameters button and select maize stem, or maize leaf, in the first drop-down box.Click the data management button, set the work directory, and import all of slice images in the directory. Then select single or multi-slice images into the image pipelines. Next, determine the pixel size of the image.Pick the method parameters button and enter the pixel size of the image in the appropriate edit item. After this, click the phenotyping computation button to automatically extract phenotypic traits of the vascular bundles for all of the selected slice images. Click the statistical analysis button to output the results of these analysis as a text or CSV file.Import the reconstructed images of maize roots, and determine the accurate space parameters. Then use the recursive gaussian tool to smooth the images, and improve image quality. Adjust the threshold parameters to conduct 3D segmentation of metaxylem vessels.This will create a uniform color label for each connected metaxylem vessel. Then, improve and identify the metaxylem vessels interactively using morphology bitwise, and flood-fill operations. Next, conduct a volume visualization and surface reconstruction of the vessels.Finally, use the mask statistics tool to count and measure the phenotypic traits of a vessel at the 2D and 3D levels. Using the analysis outlined in the protocol, the phenotypic traits of vascular bundles in maize stems, leaves, and roots were computed. A generated 3D visualization of the root and metaxylem vessels was created, using the results of segmentation, reconstruction, and volume visualization.The proper sample preparation protocol significantly improves the scanning emitting quality of micro-CT for maize stem, leaf, and root. With the automatic emit pipeline to rapidly extract the phenotypical traits of vascular bundles for maize stem and leaf. And the setup and emit processing scan to analyze the 3D phenotypic traits of vascular bundles for maize root.This technique provides a practical way for accurate and rapid phenotypic qualification and identification of maize vascular bundles.

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