Topic 19.4 Advances in Root System Phenotyping
The study of root system architecture has lagged behind other areas of plant research because of the difficulties of observing complex root systems growing in an opaque soil medium. The main limitations have been the lack of appropriate methods to non-invasively manipulate and image roots, and the lack of adequate mathematical and computational tools to describe the complex spatial structure of root systems. Recent research advances in this field have been driven by technological improvements in growth methodology, along with the simultaneous expansion of imaging and analysis techniques and germplasm resources. Laboratory growth methods used to provide access to root systems include hydroponics, agar plates, paper/cloth pouches, gel plates, box and cylinder growth systems, and aeroponic arrangements. Greenhouse growth methods typically include pots, cylinders, plates, and troughs that are filled with soil, soil substitute, or sand mixtures. Underground laboratories equipped with soil viewing windows—called rhizotrons and minirhizotrons—have also been developed to complement coring and trenching techniques in field and greenhouse settings. At the same time, root system image capture techniques have been expanded and include digital scanners and cameras, as well as methods borrowed from medical fields, including X-ray radiography, neutron radiography, laser scanning, magnetic resonance imaging (MRI), positron emission tomography (PET), computed tomography (CT), and microcomputed tomography (μCT).
Root system image capture methods include two-dimensional (2D) and three-dimensional (3D) imaging. Additionally, it is possible to reconstruct 3D images from 2D data collected via photography, scanning, X-ray radiography, and neutron radiography. 3D image capture methods involve either surface reconstruction methods or volumetric reconstruction methods. For surface reconstruction, root systems are imaged at relatively low resolutions and represented as solid objects known as surface models. 3D reconstruction techniques were initially developed for the computer vision discipline, and were later adapted for root system imaging. This type of root imaging and analysis platform is depicted in Web Figure 19.4.A. Here, rice seedlings are grown in a transparent gellan gum cylinder (see Web Figure 19.4.A, parts B and C) and the root system is imaged as shown in Web Figure 19.4.A (part A). The glass cylinder holding the rice plant is placed onto a rotational stage. The stage and digital camera are both computer controlled to enable the capture of multiple 2D images of the root system as it is rotated over a full 360° revolution. Subsequently, a 3D root reconstruction (Web Figure 19.4.A, part D) is generated from the 2D images using a custom-designed software program, and several root system architecture traits that describe different features of the root system are quantified. The high throughput potential of this approach has enabled researchers to conduct whole-genome association mapping of various root system architecture traits for 425 different rice accessions. In this way, it is now becoming possible to identify discrete regions of the plant genome that contain genes controlling specific root architecture traits.
Volumetric imaging techniques from the medical fields have also been used to image root systems. X-ray computed tomography (CT) and microcomputed tomography (μCT) use X-ray beams to non-destructively capture cross-sectional slices of root systems that are growing in soil substrates. The X-rays are emitted and captured from rotational positions around the imaging volume, allowing root images to be reconstructed and extracted from the surrounding substrate. Other, similar approaches use different medical imaging technologies, including magnetic resonance imaging (MRI) and positron emission tomography (PET), to obtain a series of 2D root images that are reconstructed into a 3D representation of root system architecture.
Web Figure 19.4.A 3D Root Growth and Imaging System. A, Diagram of 3D digital imaging system used for capturing image sequences; 40 2D images are taken every 9 degrees of rotation over a full 360° revolution. (L-lightbox; OCT-optical correction tank; IT-internal turntable; ET-external turntable; MI-magnetic interface; GC-growth cylinder; C-camera; CC-computer controlling turntable and camera). B, Growth cylinder containing gellan gum and a 10-day old Azucena rice seedling. C, Representative single 2D root system image from an image sequence captured with the 3D imaging system. D, 3D reconstruction of the rice root system showing the five root types: primary (pr), embryonic crown (ecr), postembryonic crown (pecr), large lateral (llr), and small lateral (slr) roots. The primary, crown, and large and small lateral roots can be visually distinguished from one another. (Figure A: Clark, R. T., MacCurdy, R. B, Jung J. K., Shaff, J. E., McCouch, S. R., Aneshansley D. J., and Kochian, L. V.  Three-dimensional root phenotyping with a novel imaging and software platform. Plant Physiology 156: 455–465. URL: www.plantphysiol.org. Copyright American Society of Plant Biologists.)