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

Summary

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.

Abstract

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.

Introduction

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 plants^2,3. At present, studies on the anatomical structure of vascular bundles mainly utilize microscopic and ultramicroscopic techniques t....

Protocol

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.......

Discussion

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 .......

Materials

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