Through our assessment of lab-based chamber systems, we identify unique advantages and challenges associated with each system .Lastly, we offer our perspectives on areas in which technological advances are needed to fill current knowledge gaps.In studying rhizosphere processes, the myriad of complex interactions among members of the rhizosphere are often dissected to two interacting variables such as root-and-soil or root-and-microbes, etc. Each of these interactions inherently operates under distinct parameters and requires specifically designed platforms to effectively answer different research questions. This review is structured in a way that first describes each rhizosphere process briefly and then reports on the specific growth chamber systems designed to facilitate experiments for answering related research questions. The major rhizosphere processes discussed below include root system architecture, physicochemical gradients in the soil, exudation patterns by the roots and interactions between roots and nematodes, fungi or bacteria. Root system architecture encompasses structural features that provide spatial configuration such as root length, width, spread and number and is an important rhizosphere parameter in regulating soil porosity, and nutrient and water uptake efficiency by plants . Plants have been observed to “sense” and direct root growth toward nutrient sources in soil, and the RSA of a plant exhibits great malleability in response to environmental stimuli which in turn, influences microbial communities . For instance, bean plants grew deeper roots under drought conditions to enhance water foraging capabilities while low phosphate conditions stimulated the formation of dense lateral roots involved in P uptake from upper soil layers . Given that most soils are heterogenous,black plastic nursery pots understanding the RSA of plants becomes critical in improving resource use efficiency and agricultural yields . Often, RSA in pot-grown plants is investigated by excising the roots via mechanical means such as root washing or blowing with compressed air . These methods are, however, time-consuming, cause inevitable damage of fine root hairs and result in loss of spatial and temporal information .

An appealing alternative for studying RSA is the use of rhizotrons. Rhizotrons were initially constructed as underground facilities designed for viewing and measuring roots in the field . In the lab, the rhizotron implies a chamber constructed using two vertical sheets with at least one or both of the sheets being transparent and/or removable . This allows repeated visual inspections of individual roots; a feature unachievable with destructive sampling. In some cases, the word “rhizobox” is used for a similar set up although this was first introduced in as compartmentalized systems to separate the root and soil compartments . Rhizotrons/rhizoboxes are often constructed with PVC or acrylic materials and come in many sizes to accommodate different plants with soil or soil-less substrates . Root growth and morphology in the rhizotron can be tracked by a variety of methods ranging from manual tracing onto a plastic sheet, using handheld or flatbed scanners to fully automated time-lapse imaging camera systems . Data can be subsequently analyzed with a wide range of software packages . Affordable and robust RSA imaging platforms using rhizotrons have also been developed for increased accessibility in low-income countries . The versatile construction of a rhizotron design for RSA studies has inspired many variations. For instance, ara-rhizotrons were designed to enable the study of 3D canopy competition with simultaneous root growth observation in an Arabidopsis plant population . The horizontal and radial design of HorhizotronTM and mini-Horhizotron consisting of transparent quadrants attached to a central chamber were developed to study lateral growth of roots in a semi-3D space and to perform post-transplant assessment . The separated quadrants can also be used with different soil substrates simultaneously to study substrate effects on root growth . A rhizotron fitted with water-tight gasket seals has also been used successfully to investigate the RSA of plants under water-logged conditions . Despite the continuous real-time visual read-out, most rhizotron designs suffer from inevitable loss of information from roots occluded by soil particles. The GLO-Roots system overcomes this by imaging from both sides of the rhizotron while using bio-luminescent roots to create higher contrast against the soil, enabling quantitative studies on RSA . Following advances in engineering and device fabrication, more rhizotron variants adapted to specific plant growth conditions can be envisioned. In a typical topsoil, approximately half is composed of solid minerals and organic matter while the rest is a fluctuating composition of water and gas filled spaces influenced by environmental conditions and uptake/release of solutes from plants . Changes in gaseous and hydrologic parameters, such as ions, O2 and moisture among others, create a spatially complex environment that influences microbial communities and overall plant health.

These physicochemical fluxes are heterogeneously distributed along roots and vary with root types and zones . Often, they exist as gradients in the rhizosphere , thus emphasizing the need for non-destructive sampling in order to accurately capture processes occurring at biologically relevant times and scales. Rhizotron chambers with a visually accessible rhizosphere allows in situ and continuous mapping of these gradients in the soil through the use of different types of imaging methods. For instance, photo luminescence-based optical sensors enable in situ, repeated detection of small molecule analytes in addition to pH , O2 and NH4 . Methods like zymography to detect enzyme activity and diffusive gradients in thin film can be used to map solute concentrations in the soil down to sub-mm scales with high spatial resolution more realistically than traditional destructive approaches. For example, transport and distribution of water in the rhizosphere soil has been imaged on both 2D and 3D planes by coupling a rhizotron with neutron radiography and tomography, respectively and showed varying moisture gradients along the root system with higher water uptake at the rhizosphere compared to bulk soil. On the other hand, if the rhizotron slabs are thin enough , even simple imaging solutions based on light transmission can be set up to capture water uptake by roots in sand . Despite trade-offs in method sensitivity between these two studies, a rhizotron set up is critical in both designs and illustrates its adaptability to multiple equipment. Roots exude a substantial amount of photosynthetically fixed organic carbon into the soil consisting of a wide variety of compounds such as sugars, organic acids, and primary and secondary metabolites . Together with mucilage and border cells , root exudates provide a major source of nutrients for the rhizosphere microbiome . Root exudation is regulated under genetic control  as well as in response to environmental conditions in the soil such as nutrient limitations or increase in toxicity . Exudate patterns are also recognized as one of the strongest drivers shaping the rhizosphere microbiome . As a central player in the rhizosphere ecosystem, it is imperative to understand root exudation patterns to unravel subsequent impacts to the surrounding soil and microbial community. Improvements in analytical instrumentation have made it possible to move from targeted to untargeted explorations with mass spectrometry to create root exudate fingerprints in its entire complexity . Regardless, the impact of such techniques relies partly on our exudate sampling techniques.

Detection of exudates in real-time is difficult due to rapid bio-transformation and sorption to the soil matrix. As such, common collection methods rely on root washing in hydroponic systems to overcome complications in the soil matrix and preserve native exudation profiles. However, a comparison between a soil-based collection method and hydroponic methods showed varied responses particularly in amino acid exudation although the underlying cause was not elucidated . It is possible that the differing growth conditions between hydroponics and soil,greenhouse pot which include differences in gas concentrations, mechanical impedance and microbial spatial composition, can elicit differing root exudation responses to the same environmental stimuli. Rhizoboxes offer the advantage of localized sampling in soil using sorption media such as paper and membrane filters, compound specific ion exchange binding resin or micro-suction cups placed closed to root zones of interest to collect exudates . Moreover, in a rhizobox fitted at the bottom with a porous rootimpenetrable membrane, a root mat is allowed to be formed which is then further transferred onto a collection compartment . The collection compartment containing soil could then be cut into thin slices parallel to the membrane to represent differing distances from the rhizosphere . While this approach can be used to investigate exudate release and sorption under soil conditions, the root mat growth generalizes exudate production in terms of the whole root system and occludes spatial exudation patterns. In a hybrid set up by Oburger et al. , the rhizobox is transplanted to a second specialized rhizobox for continued vertical root growth. This specialized rhizobox consists of a nylon membrane close to the transparent side to restrict root growth into the soil except for root hairs . This creates a vertical flat root mat onto which localized exudate samples can be collected. A comparison of this novel set up to conventional collection methods showed that amino acid exudation rates were most varied among the different methods , further highlighting the need for specialized chambers. Nonetheless, successful implementation of these chambers is still limited to fast-growing plants which can form active root mats. The high density of root mats could also lead to unnatural root exudate levels and an overestimation of rhizosphere effects. In addition, care has to be given to the choice of membrane as selective sorption of certain root exudates onto the membrane may also occur . Free-living nematodes are ubiquitous in the soil. They are beneficial to the plants by playing a role in nutrient cycling and in defense against insects and microbial infections through signaling interactions with the roots . Conversely, infections by parasitic nematodes in the roots increase the plant’s susceptibility to stress and other pathogenic bacteria, fungi, and viruses creating major losses in crop productivity . With an impending rise in nematode infections due to climate change, understanding nematode behavior and interactions in the rhizosphere becomes important to develop appropriate bio-control methods to ensure long term food security .

Traditional nematode studies are performed in petri dishes with agar or culture media . However, these substrates do not accurately emulate the physical textures and heterogeneity of soil and create homogenous solute and temperature gradients which could impact nematode behavior and interactions with the roots . Indeed, nematode motility speed and dispersal decreased in substrates more closely mimicking sand . On the other hand, studying nematode behavior in the soil is a difficult endeavor as its near-transparent body and small size makes it almost indistinguishable from soil particles. Cross-sectioning and staining infected roots make it possible for nematode visualization but they are destructive and provide only static snapshots of cellular changes or nematode behavior during infections . On the other hand, microscopy rhizosphere chambers provide non-invasive detection and observation of nematode activity in the rhizosphere . The roots in these chambers grow between a glass slide and a nylon membrane . The membrane restricts movement of roots except root hairs into the soil while the transparent glass enables microscopy of the roots at high resolution . Coupled with fluorescently stained nematodes, microscopy rhizosphere chambers allowed for non-destructive in situ observations of nematode infection in its host species over the entire life of the parasite . Nonetheless, staining nematodes is an additional challenge as nematode cuticles are impermeable to stains . This can, however, be alleviated by using advanced imaging technologies which eliminates the need for staining. A recent study demonstrated live screening of nematode-root interactions in a transparent soil-like substrate through the use of label-free light sheet imaging termed Bio-speckle Selective Plane Illumination Microscopy coupled with Confocal Laser Scanning Microscopy . Using this set up, researchers were able to monitor roots for nematode activity at high resolution and suggest its possible use in rapid testing of chemical control agents against parasitic nematodes in soil-like conditions . Fungal communities in the rhizosphere are involved in the degradation of organic matter in the soil and subsequent nutrient turnover affecting plant health as well as the microbial community . Fungal biomass often reaches a third of total microbial biomass carbon and almost all terrestrial plants are able to form symbiotic associations with mycorrhizal fungi .