Rhizoslides: paper-based growth system for non-destructive, high throughput phenotyping of root development by means of image analysis

Abstract

BackgroundA quantitative characterization of root system architecture is currently being attempted for various reasons. Non-destructive, rapid analyses of root system architecture are difficult to perform due to the hidden nature of the root. Hence, improved methods to measure root architecture are necessary to support knowledge-based plant breeding and to analyse root growth responses to environmental changes. Here, we report on the development of a novel method to reveal growth and architecture of maize root systems.ResultsThe method is based on the cultivation of different root types within several layers of two-dimensional, large (50 × 60 cm) plates (rhizoslides). A central plexiglass screen stabilizes the system and is covered on both sides with germination paper providing water and nutrients for the developing root, followed by a transparent cover foil to prevent the roots from falling dry and to stabilize the system. The embryonic roots grow hidden between a Plexiglas surface and paper, whereas crown roots grow visible between paper and the transparent cover. Long cultivation with good image quality up to 20 days (four fully developed leaves) was enhanced by suppressing fungi with a fungicide. Based on hyperspectral microscopy imaging, the quality of different germination papers was tested and three provided sufficient contrast to distinguish between roots and background (segmentation). Illumination, image acquisition and segmentation were optimised to facilitate efficient root image analysis. Several software packages were evaluated with regard to their precision and the time investment needed to measure root system architecture. The software 'Smart Root’ allowed precise evaluation of root development but needed substantial user interference. 'GiaRoots’ provided the best segmentation method for batch processing in combination with a good analysis of global root characteristics but overestimated root length due to thinning artefacts. 'WhinRhizo’ offered the most rapid and precise evaluation of root lengths in diameter classes, but had weaknesses with respect to image segmentation and analysis of root system architecture.ConclusionA new technique has been established for non-destructive root growth studies and quantification of architectural traits beyond seedlings stages. However, automation of the scanning process and appropriate software remains the bottleneck for high throughput analysis.

Accelerating genetic gains in legumes for the development of prosperous smallholder agriculture: integrating genomics, phenotyping, systems modelling and agronomy

“…Seeds are planted on the slit of the Plexiglas • Allows separation of crown roots from embryonic roots Le Marié et al (2014) Shovelomics (PennState Colleage of Agriculture, USA)…”

Quantitative 3D Analysis of Plant Roots Growing in Soil Using Magnetic Resonance Imaging

“…Methods in which the whole root system is visible are typically based on artificial media such as paper pouches (Chen et al, 2011;Le Marié et al, 2014), three-dimensional (3D) gels (Iyer-Pascuzzi et al, 2010), and hydro-or aeroponics (Herdel et al, 2001). Results may thus not be directly transferable to plants grown in natural 3D soil environments (Gregory et al, 2003).…”

Two-dimensional root phenotyping system based on root growth on black filter paper and recirculation micro-irrigation

“…The system is thus especially suitable for evaluating dynamic changes in RSA of different genotypes in response to environmental conditions. A similar system of so called "rhizoslides" was developed by Le Marié et al (2014). Rhizoslides utilize Figure 2.…”

“…Additionally, the black filter paper presents a marked contrast to the roots, which simplifies an image analysis, whether the images were taken with digital SLR camera or flatbed scanner. The main advantage of rhizoslides is the ability to separate and evaluate the crown and seminal roots in maize (Le Marié et al 2014). It is thus evident that each system has its advantages for certain specific applications.…”

“…However, these methodologies still had numerous limitations such as root size restrictions or laborious access and measurement of root system. One of the most promising non-soil systems for root phenotyping was recently developed on the basis of growing roots on vertically positioned germination paper closed between two acrylic sheets (Le Marié et al 2014).…”