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Abstract

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Biology

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

Published: October 9th, 2018

DOI:

10.3791/58501

1Beijing Key Lab of Digital Plant, Beijing Research Center for Information Technology in Agriculture, Beijing Academy of Agriculture and Forestry Sciences
* These authors contributed equally

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 '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 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 t....

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1. Sample Preparation Protocol

  1. 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.......

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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 µm/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.......

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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 .......

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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).

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Name Company Catalog Number Comments
Skyscan 1172 X-ray computed tomography system Bruker Corporation, Belgium NA For CT scanning
CO2 critical point drying system (Leica CPD300) Leica Corporation, Germany NA For sample drying
Ethanol Any NA For FAA fixation
Formaldehyde Any NA For FAA fixation
Acetic acid Any NA For FAA fixation
Surgical blade Any NA For cutting the sample sgements
3D printer Makerbot replicator 2, MakerBot Industries, USA NA For printing the sample baskets of maize root, stem, and leaf
Centrifuge tube Corning, USA NA Place the root, stem, or leaf materials
Solid iodine Any NA For sample dyeing
SkyScan Nrecon software SkyScan NRecon, Version: 1.6.9.4, Bruker Corporation, Belgium NA For image reconstruction
VesselParser software VesselParser, Version: 3.0, National Engineering Research Center for Information Technology in Agriculture (NERCITA), Beijing, China NA Image analysis protocol for single CT image of maize stem or leaf
ScanIP ScanIP, Version: 7.0; Simpleware, Exeter, UK NA 3D image processing software
Latex gloves Any NA
Tweezers Any NA

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