Understanding these intricate rhizosphere relationships is vital in devising strategies to increase plant productivity and comprehend localized biogeochemical processes. In many rhizosphere studies, the use of pots and containers is predominant as it allows the plants to be cultivated under controlled conditions and at low cost. Compared to field studies, growth of plants in defined spaces also offers advantages in ease of handling, monitoring and sampling . Much of what we know of the rhizosphere microbiome has resulted from such pot-grown plants. However, since the rhizosphere and roots are still out of view in the soil, destructive sampling of the root is required prior to analysis. Destructive sampling may result in the loss of three-dimensional spatial information on rhizosphere processes over time, which is increasingly being recognized as a critical parameter. On the other hand, soil free techniques such as hydroponics and aeroponics can provide visual access to the rhizosphere circumventing the need for destructive sampling. Other alternatives are gel-based substrates which can maintain rhizosphere transparency as well as the 3D architecture of roots and have been applied successfully in high throughput imaging, phenotyping and trait mapping platforms . Nonetheless, the root phenotype and traits of plants grown under soil-free conditions are known to differ from those of soil-grown plants . These soil substitutes do not also accurately simulate the heterogeneous nature of soil aggregates, thus complicating extrapolations for field relevance. Sophisticated imaging approaches such as magnetic resonance imaging and X-ray computer tomography can be used to analyze root systems in the soil with minimal disturbance but they are low throughput,led grow lights expensive and may not be easily accessible . It is apparent that structural changes in design catered to solving specific challenges in the rhizosphere are indeed necessary. To overcome these challenges relating to the rhizosphere in soil, specialized plant growth chamber systems have been designed, and successful implementation has led to multiple variations of similar designs.
These specialized systems often have a visible rhizosphere which enables coupling with other technologies thereby increasing the breadth of experimental techniques applicable to the rhizosphere system. This review discusses representative growth chamber systems designed to study major rhizosphere processes and interactions in soil. Growth platforms resembling conventional containers such as pots and tubes are not covered. specifically, the reviewed growth systems are selected based on the following criteria: the growth chamber is amenable for use with soil/soil-like substrates and therefore, hydroponics, aeroponics and agar/gel-based systems are not discussed except in microfluidic-based platforms, it is built with the intention to maintain growth of the plant and has architectural features distinct from conventional pots, and lastly it is able to be set up in a laboratory; i.e., field measurement systems and observation platforms are not included. For instance, a minirhizotron, consisting of a camera mounted in a glass tube submerged in the soil which provides non-destructive root imaging over time will not be discussed as it is out of the scope of this review. Through our assessment of lab-based chamber systems, we identify unique advantages and challenges associated with each system . We hope that future fabrication designs can benefit and improve on designs that work well. 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, 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 bioluminescent 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,vertical grow system 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.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 biotransformation 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, 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 root impenetrable 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 .