Three-dimensional root phenotyping with a novel imaging and software platform

Abstract

A novel imaging and software platform was developed for the high-throughput phenotyping of three-dimensional root traits during seedling development. To demonstrate the platform's capacity, plants of two rice (Oryza sativa) genotypes, Azucena and IR64, were grown in a transparent gellan gum system and imaged daily for 10 d. Rotational image sequences consisting of 40 two-dimensional images were captured using an optically corrected digital imaging system. Three-dimensional root reconstructions were generated and analyzed using a custom-designed software, RootReader3D. Using the automated and interactive capabilities of RootReader3D, five rice root types were classified and 27 phenotypic root traits were measured to characterize these two genotypes. Where possible, measurements from the three-dimensional platform were validated and were highly correlated with conventional two-dimensional measurements. When comparing gellan gum-grown plants with those grown under hydroponic and sand culture, significant differences were detected in morphological root traits (P < 0.05). This highly flexible platform provides the capacity to measure root traits with a high degree of spatial and temporal resolution and will facilitate novel investigations into the development of entire root systems or selected components of root systems. In combination with the extensive genetic resources that are now available, this platform will be a powerful resource to further explore the molecular and genetic determinants of root system architecture.

Figure 1.

3D root growth and imaging system. A, Schematic of the 3D imaging system used for capturing image sequences consisting of 40 2D images every 9° of rotation over a full 360° revolution. C, Camera; CC, computer controlling turntable and camera; ET, external turntable; GC, growth cylinder; IT, internal turntable; L, light box; MI, magnetic interface; OCT, optical correction tank. B, Growth cylinder containing gellan gum and a 10-d-old Azucena rice seedling. C, Representative single 2D root system image from an image sequence captured with the 3D imaging system. [See online article for color version of this figure.]

Figure 2.

RootReader3D screen shot. RootReader3D software generates high-resolution 3D root system reconstructions from 40 2D images of root systems for plants grown in the gellan gum growth system and also provides the tools to perform both automated and semiautomated trait analysis. The screen shot shows the RootReader3D toolbar (top), the root model volume window (left), and the reconstruction volume window (right) used for visualizing and interacting with the generated 3D root system models. The shaded slice through the 3D root model (left) corresponds to the horizontal cross-section through the root system shown in the reconstruction volume window on the right. [See online article for color version of this figure.]

Figure 3.

Rice root types. A, Depiction of the five root types: the primary (pr), embryonic crown (ecr), postembryonic crown (pecr), large lateral (llr), and small lateral (slr) roots. As labeled on the 3D root model above, the primary, crown, and large and small lateral roots can be visually distinguished from one another. Temporal imaging is performed to further separate the crown roots into embryonic and postembryonic crown root types based on emergence time. Roots that emerged from the crown between 1 and 5 d after planting were classified as embryonic crown roots, whereas roots that emerged later than 5 d after planting were classified as postembryonic crown roots. B, Average root emergence times of primary and crown roots. Roots were individually selected and measured daily to determine emergence times based on average growth rates. Error bars represent se for all roots of a particular genotype and type. [See online article for color version of this figure.]

Figure 4.

3D root system models generated from daily imaging of root systems over a 10-d period using the RootReader3D software (day 1 [D1]–day 10 [D10]). The skeletons of the root systems are shown in red, and the primary and crown roots are shown in yellow. The primary and crown roots were selected and labeled, allowing for dynamic tracking of root type-specific growth features.

Figure 5.

Quantitative description of Azucena and IR64 root system differences. A, Day-10 Azucena 3D root system reconstruction. B, Day-10 IR64 3D root system reconstruction. C, Average root length for different root types for Azucena (blue) and IR64 (red) plants, day 1 (D1) to day 10 (D10); n = 7. D, A subset of quantified RSA traits, vertical centroid position, volume distribution, and bushiness, that quantitatively describe the differences between Azucena and IR64 RSA. Each data point represents a single measurement made on an individual Azucena (blue diamonds) or IR64 (red triangles) plant on a given day. Asterisks indicate where significant differences were detected between Azucena and IR64 genotypes using a t test (P < 0.05). Volume distribution and bushiness traits were adapted to 3D from 2D methods described by Iyer-Pascuzzi et al. (2010).

Figure 6.

Root circumnutation. A, Top view of a 10-d Azucena root system reconstruction showing tangential angles (ρ_init and ρ_fin) and root segment length (L) used for measuring circumnutation. Yellow lines are the selected primary and crown roots. B, Average circumnutation rates for different root types for Azucena and IR64 genotypes. Root types are abbreviated as pr (primary root), ecr (embryonic crown roots), pecr (postembryonic crown roots), and llr (large lateral roots). Error bars represent se for all roots of a particular genotype and type.

Figure 7.

Root gravitropism. A, Depiction of a rice seedling with tangential angles (θ_init and θ_fin) and root segment length (L) used for measuring gravitropic traits. B, Average root initiation angle (θ_init) separated by root type. Root types are abbreviated as pr (primary root), ecr (embryonic crown roots), pecr (postembryonic crown roots), and llr (large lateral roots). C, Gravitropic responses for different root types. Error bars represent se for all roots of a particular genotype and type. Asterisks indicate where significant differences were detected between Azucena and IR64 genotypes using a t test (P < 0.05). [See online article for color version of this figure.]