Background

The understanding of root development and its surrounding environment, the rhizosphere, under varying conditions is crucial for optimizing plant growth and yield for agriculture. The study of plant physiological processes in response to their environment is possible with fabricated ecosystems (EcoFAB 2.0 system). This system allows high-resolution imaging of roots in a multi-factorial, controlled environment [1,2] and the characterization of shoot growth. EcoFABs enable the study of plant–microbiome interactions by introducing microbial, fungal, or bacterial cultures into the chamber, which can be easily sterilized. During experiments, the nutrient media can be replaced or altered at any time. Imaging or scanning can be conducted to collect data on root size, root hairs, hyphae, leaves, and interparticle space. Root architecture and leaf growth can be analyzed simultaneously. By imposing different conditions on the plants, we can observe which ones encourage or hinder their development.

The development of the EcoFAB system, a repeatable and versatile tool for studying plants in controlled environments, enables a more effective transfer of knowledge across different scientific communities. A standardized sterilization and germination protocol for various plant species is necessary to ensure uniform plant growth stages suitable for transfer into the sterilized EcoFAB system. Seed priming allows the control of seed hydration and induces physiological, biochemical, and molecular alterations that trigger germination [3]. With this type of treatment, it is possible to increase vigor and germination rate or reduce the time needed to obtain better seedlings. Radicle protrusion through the seed coat is the main event to observe homogeneous germination. To enhance germination, many invasive methods [4,5] have been described in the literature, mainly based on water uptake by seeds. This is why we developed a user-friendly seed treatment and germination protocol for different plant species to achieve a homogeneous germination rate and growth suitable for experiments in the EcoFAB system.

Several other tools are available to study plants and their interactions with soil, microorganisms, and ecosystems. Systems like rhizoboxes and rhizotrons [6] were initially created to study root growth in mature plants. These systems enable plants to grow in their natural soil environment. Using a non-destructive method, root or shoot growth can be assessed through successive measurements on the same plants. This approach reduces phenotypic variability linked to differences between individual plants and enhances accuracy compared to methods requiring plant destruction during sampling [7]. Recently, the CD-Rhizotron system [8] was developed to study the spatial differentiation of roots linked to different microbial communities or exudates. Fine root studies are also possible with Minirhizotrons [9] or EnRoot [10] devices, but as with previous methods, the ecosystem is not controlled, and plants remain in the field. More recent technologies, such as TRIS [11] and root arrays [12], have been developed to achieve a precise understanding of root behavior in microfluidic devices, imaging them in real-time and enabling genotyping analysis. A key limitation of these devices is their restriction to experiments on small, young plants. Additionally, these systems do not replicate field-like environments.

In contrast, EcoFABs are fully controllable hydroponics systems, offering many possibilities for experiments. A deep understanding of plant growth mechanisms and interactions has numerous direct applications in agriculture, such as improving yield and triggering some diseases caused by fungi or bacteria. Conservation efforts for endangered species are another important application, as the International Union for Conservation of Nature (IUCN) red list encourages the scientific community to engage in conservation efforts for these species. Early developmental stages are crucial for the conservation of endangered species, and EcoFABs could help determine key features of early development, particularly by optimizing life cycles and seed germination. Beyond these applications, studying plant–microbiome–soil interactions in systems like the EcoFAB holds significant potential for space exploration. As part of the Plants 4 Space Program, this research explores the use of EcoFABs in optimizing fruit and vegetable production for long-term space missions. A deep understanding of root cells and rhizosphere interactions is essential for optimizing crops in space [13], especially fresh food production to counteract the degradation of packaged food and the potential digestive issues caused by a lack of fruits and vegetables in astronauts’ diets [14]. This work introduces the potential use of strawberries [15] and lettuce in EcoFAB 2.0, aligning with space programs that have mainly focused on these crops. It also opens the door to studying additional plants in the EcoFAB system.

Inspired by the Brachypodium distachyon protocol [16,17], we present a precise germination and setup protocol in EcoFABs for Arabidopsis thaliana with the aim of extending the study of this model plant. Lettuce is widely studied in hydroponic systems for vegetable production [18,19]. Here, we developed a new approach to studying lettuce (Lactuca sativa; Outredgeous) in its early stages of development. We also propose a quite similar protocol applicable for Fragaria vesca (Hawaii 4). Our protocol can be easily adapted to many other species, including endangered ones. The main goal is to extend EcoFAB 2.0's applicability to additional plant species and provide standardized procedures that improve both the quantitative (germination rate) and qualitative (uniformity) aspects of plant germination.