Introduction

Microfluidics[1], and other engineered environments[5], [6] can produce highly controlled micrometer-scale environments for the study of organismal model systems (e.g., mammalian cells). However, scientists or engineers interested in manipulating the environment of cm-scale organisms (e.g., plants) have remarkably few convenient tools at their disposal[7], [8]. This paucity is partly due to the demanding design requirements associated with larger scales (e.g., cost). This liability is particularly evident in the study of plants and their root systems.

The development of plants in soil is an important subject of investigation. The provision of food to the global human population is under severe pressure (our supply of food is predicted to be far below demand by 2050[9]) and depends on plant roots[10] (97.6% of global calorie consumption is derived from plants[11]). Roots influence a plant's yield and whether a plant will survive stresses. We know that root growth is strongly affected by its environment, soil, but our mechanistic understanding of these effects is imperfect[10], [12] and strongly limited by technical challenges.

Root development is a difficult process to study experimentally. (i) Plants display highly variable root systems, even when genetically identical[13]. (ii) Roots are remarkably sensitive to a variety of stimuli (e.g., gravity, light, touch, moisture, nutrients, oxygen, temperature, trauma, electric fields[14]). (iii) Any volume of soil is unique and impossible to replicate exactly[15], [16]. (iv) Its heterogeneity makes it opaque to most forms of radiation[17]. (v) Its structural and chemical characteristics (i.e., porosity, surface chemistry, nutrient gradients, oxygen gradients, bulk composition, soil biota) cannot be independently manipulated.

One approach to avoid this complexity is to characterize the growth of plants in soil-less media, e.g., hydrogels, paper, glass beads, sand. These systems are less inhomogeneous and irreproducible than soil and can be modified – usually to a limited extent – to mimic soil properties such as chemical composition [18], physical structure [19], [20], water availability [21], refractive index [22], or mechanical strength [23]. However, the lack of modularity, versatility, structural precision, and the very limited control over structural and chemical heterogeneities in these systems severely limits the type, complexity, and reproducibility of the experiments they can perform. Microfluidic approaches offer fascinating capabilities for the study of plant roots, but are subjected to limitations in their throughput and in the size of the plants they can host [4], [24], [25].

We here demonstrate that LEGO bricks are highly convenient and versatile building blocks for building cm-scale engineered environments for plant roots. Their modularity enables the fabrication of environments with highly controlled structural and chemical heterogeneities that are suitable for convenient quantitative studies of environmental effects on plant phenotypes[26].

System Design

A convenient experimental platform for the study of root development in controlled environments must satisfy a demanding set of design constraints. LEGO bricks, while conceived and sold as toys, satisfy these constraints.

High throughput

The ability to run a large number of experiments at the same time is essential for the establishment, for example, of genotype-environment-phenotype relationships[32]. A LEGO structure like the one shown in Figure 1 can be assembled in less than a minute.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 1. Scheme of the process of carrying out a plant growth experiment using LEGO bricks as building blocks.

The same process can be used to prototype and fabricate other biological experiments.

https://doi.org/10.1371/journal.pone.0100867.g001

Results and Discussion

Figure 1 shows a flow diagram of the design, assembly, disassembly, and re-assembly of an experiment based on LEGO bricks. The website of the LEGO Group (www.lego.com) provides a free software (LEGO Digital Designer, LDD) for the CAD-like design of structures using any available LEGO brick. The software outputs a step-by-step assembly guide and a list of the required parts. Individual bricks can be purchased through the “Pick a Brick” section of www.lego.com or other outlets (e.g. local LEGO stores, EBay). Sterilization of LEGO bricks can be performed before or after assembly. The preservation of sterility requires the structure to be maintained in a sterile container during the course of an experiment.

The simplest example of a plant germination and growth environment based on LEGO bricks is shown in Figure 1. The LEGO bricks are assembled into a container that contains a root growth medium on which a seed is germinated and grown: Figure 1, for example, shows a Brassica rapa, Wisconsin Fast Plant, Astroplant, dwf1, growing on a transparent hydrogel, Gellan gum. While gel media for root growth are very commonly used in experiments[26], they are not the best mimic of soil: root architectures grown in an homogeneous media will not match those of plants grown in real soil[36]. However, gel media allows us to demonstrate three essential capabilities of LEGO-based biological environments: their ability to hold liquids, their compatibility with real-time observation and root structure analysis, and their use in generating reconfigurable environments that include controlled heterogeneities. Furthermore, LEGO environments are not limited to gel media: the environment shown in Figure 1 can hold other media of choice, e.g., sand, perlite, soil.

Since structures built from LEGO bricks are not waterproof, their use to hold gels requires some stratagems (see Supporting Information for details and Movie S1 for a demonstration). The LEGO structure must be chilled in a freezer before the cool gel solution is poured in it just prior to setting. Using this approach, leakage of the gel solution was minimal. These basic environments can be easily scaled to match the dimensions of the organism under consideration and the time the organism is allowed to grow. Figures 2a, 2b, and 2c show the use of LEGO bricks to create containers with very different dimensions (5×5×5 cm, 10×10×5 cm, and 20×20×10 cm) for the growth of Fast Plants, Triticum polonicum (Wheat), and Zea mays (Corn).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 2. Versatility, transparency, and modularity of the LEGO-based environments for plant growth.

a-c) pictures of basic LEGO-based environments growing Fast Plants, Wheat and Corn. The size of the environments can be controlled to match the size of the organism under consideration. d) Timelapse imaging of Lepidium sativum root development through the walls of a LEGO-based environment. The images indicate the time since germination. e) Examples of a LEGO-based system that allows for the dynamic change of the environment of a plant. Two plants (Fast Plants) are grown in isolated environments. The environment is then modified, during growth, to allow the two plants to share the same environment and interact.

https://doi.org/10.1371/journal.pone.0100867.g002

The transparency and flat walls of LEGO bricks allows for good quality real time imaging of the development of the root system. Figure 2d shows time-lapse imaging of Lepidium sativum (Garden cress) roots over the course of ∼48 hrs from germination in a LEGO-based environment. The plant was chosen for its relatively fine roots (∼350 µm thickness) that would have been hard to image in a poorly transparent system.

The reversible nature of the mechanical bond between the bricks provides two important capabilities: the creation of reconfigurable biological environments, and of highly controlled heterogeneities (i.e., solid obstacles, air pockets, and chemical and soil biota gradients) in an otherwise homogeneous growth medium. Figure 2e demonstrates a reconfigurable plant growth environment. Two Fast Plants were grown in gel in separate containers assembled on the same base plate. The LEGO brick walls separating the two containers were removed and reconfigured to make one larger container. The volume separating the two plants was then filled with more gel, fluidically connecting the two plants. Figure 3 demonstrates the generation of controlled heterogeneities in a homogeneous gel medium for plant growth by a simple templating strategy borrowed from the materials science “toolbox”. A gelling mixture was poured into a LEGO-based mold. LEGO-based features in the mold can be used as solid heterogeneities to study the physical interaction of plant roots with solid objects (thigmotropism). After gelation, LEGO-based molds could be removed, leaving behind precisely positioned air pockets that would serve as sources of oxygen gradients into the gel. These pockets could be then refilled with a hydrogel containing a desired chemical to generate precisely positioned one-dimensional (Figure 3, bottom left panel) or two-dimensional (Figure 3, bottom right panel) nutrient gradients. The above process can be combined to create environments with solid heterogeneities, air pockets (i.e., oxygen gradients), and chemical (e.g., nutrients, toxins, signaling molecules) gradients simultaneously (see Appendices S1).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 3. Fabrication of controlled heterogeneities in plant growth environments.

Sequence of diagrams and corresponding images illustrating the generation of a 1D and 2D heterogeneities (solid features, air pockets, and chemical gradients) across a developing root system of a Fast Plant. In the bottom panels, the red linear gradient is of MS nutrients (dye is added for visibility), while the radial gradients are from potassium phosphate (green), potassium nitrate (yellow), calcium chloride (red), and magnesium sulfate (blue).

https://doi.org/10.1371/journal.pone.0100867.g003

Conclusions

In summary we demonstrated that LEGO-based environments can (i) scale to the size of the organism under consideration, (ii) allow for real time monitoring of root systems in 3D, (iii) be structurally reconfigured to change the environment of an organism during its development, and (iv) generate precisely controlled heterogeneities (i.e., solid barriers, air pockets, chemical and soil biota gradients) in an otherwise homogeneous growing medium.

This manuscript also proposes a broader concept: the use of reusable and mechanically interlocking building blocks for the construction of biological environments for cm-scale organisms and systems of organisms. Modular and reusable building blocks can alleviate the challenges associated with the large scales of plant science experiments, while providing new capabilities (e.g., controlled heterogeneities, reconfigurable environments) for the study of environmental effects on biosystem development. Furthermore, this concept provides materials chemists and engineers with two stimulating opportunities: (i) to creatively engage with the synthesis or development of increasingly capable cm-scale biological environments for important organisms such as plants, and (ii) to use these environments to test hypothesis concerning plants that are compatible with their skillset. Compelling opportunities lie in extending our approach to chemically synthesized bricks, LEGO-compatible 3D-printed bricks and objects, and commercial bricks from other manufacturers. Our laboratory will be introducing a set of integrated tools for the fabrication of frugal but sophisticated[37] cm-scale environments for the study of plants and other organisms[35].

Supporting Information

Acknowledgments

We thank Dr. Kuloth V. Shajesh for valuable discussions and William Rekemeyer for help in the laboratory.