Hydrogel-based transparent soils for root phenotyping in vivo

Abstract

Root phenotypes are increasingly explored as predictors of crop performance but are still challenging to characterize. Media that mimic field conditions (e.g., soil, sand) are opaque to most forms of radiation, while transparent media do not provide field-relevant growing conditions and phenotypes. We describe here a "transparent soil" formed by the spherification of hydrogels of biopolymers. It is specifically designed to support root growth in the presence of air, water, and nutrients, and allows the time-resolved phenotyping of roots in vivo by both photography and microscopy. The roots developed by soybean plants in this medium are significantly more similar to those developed in real soil than those developed in hydroponic conditions and do not show signs of hypoxia. Lastly, we show that the granular nature and tunable properties of these hydrogel beads can be leveraged to investigate the response of roots to gradients in water availability and soil stiffness.

Keywords: hydrogels; microbiome; plants; soil; transparent.

Fig. 1.

Fabrication and physical properties of hydrogel-based TS. (A) Sketch of the spherification process to make the hydrogel beads. (B) TS before (Left) and after (Right) saturation with nutrient growth media [0.5× Murashige and Skoog medium (Top), lysogeny broth (Middle), and soil extract (Bottom)]. The logo behind the cuvette is not visible before saturation but becomes clearly visible upon saturation of the TS. (C) Transmittance of TS (at 1,080 nm, in 0.5× MS) as a function of the concentration of the polymer and MgCl₂ solutions used during spherification. The colormap also shows the length of the optical path that leads to 10% transmittance at 1,080 nm. (D) Collapse stress of TS (filled with 0.5× MS) as a function of the concentration of the polymer and MgCl₂ solutions used during spherification. The colormap also shows the thickness of TS that would collapse at its bottom. (E) Bead size as a function of the inner diameter of the nozzle used during spherification. (F) Total and effective porosity of TS as a function of the size of the beads. (G) Shrinkage of the beads as a function of time (with and without plants) and their recovery upon saturation with media on days 7 and 14.

Fig. 2.

Root phenotyping in TS. (A) Time lapse (24-h interval) in vivo root phenotyping of G. max growing in hydroponic conditions (Top) and in TS (Bottom) between day 9 and day 17 from germination. Soil extract was used as a nutrient medium for both treatments. (B–D) Comparisons of the temporal evolutions of root phenotypes (n = 5) for the TS (blue) and hydroponic (gray) treatments: B plots the ratio between the length of the side roots and main root, and shows accelerated development of the side roots in hydroponic conditions; C plots the ratio between the surface area and the convex area of the root system, and shows increased space-filling of the TS-grown roots; and D plots the average root diameter, and shows larger diameter of for the TS-grown roots.

Fig. 3.

Root phenotyping in TS. (A and B) Comparison of (A) G. max roots and (B) their mechanical stiffness, after growth in hydroponics (Left), TS (Center), and sterilized field soil (Right). (Scale bar in B, 1 cm.) (C) Comparison of biomass, root morphology traits, and gene expression (Glyma.11G121800 and Glyma.06G070500) in G. max plants grown in hydroponics, TS, and sterilized field soil. Error bars for biomass and root morphology traits indicate SD (n = 9); error bars for gene expression represent SD (n = 4). Different letters above the histograms (a, b, c) indicate significant differences between treatments (P < 0.05, one-way ANOVA followed by Tukey’s test).

Fig. 4.

Applications of TS. (A) Sketch of the in vivo phenotyping of A. thaliana roots in TS in a modified Petri dish. (B) In vivo confocal microscopy of A. thaliana roots at different depths into the TS [0 mm (Left), 2.4 mm (Right)]. (C) In vivo fluorescence microscopy at different depths into the TS [0 mm (Left), showing the surface of a TS bead; 2.9 mm (Right), showing visible and fluorescence overlay of the A. thaliana root behind the bead]. (D) In vivo pH mapping in TS obtained by overlaying a root image (obtained in the NIR from a G. max plant) with a yellow colormap obtained from a visible photograph of the same root system after isolating the color change caused in a pH indicator in the TS by acidification. (E) Growth of a G. max root system in a TS medium with a graded water potential. The left half of the TS medium contains 200 g⋅L⁻¹ PEG (−5.11 bar water potential), while the right half contains TS without PEG (0 bar water potential). The root develops significantly (P = 10⁻¹¹, n = 10; different letters above the histogram indicate significant differences) toward the region of higher water availability. (F) Growth of a G. max root system in a TS medium with graded stiffness (collapse stresses of 17.12, 23.52, and 55.52 kPa from the top down; different letters above the histogram indicate significant differences). The graded TS leads to the development of a shallower root system with a significantly higher fraction of projected root area (P < 0.001) in the top layer of soil, compared with a homogeneous control.