Design of a comprehensive microfluidic and microscopic toolbox for the ultra-wide spatio-temporal study of plant protoplasts development and physiology

Abstract

Background Plant protoplasts are basic plant cells units in which the pecto-cellulosic cell wall has been removed, but the plasma membrane is intact. One of the main features of plant cells is their strong plasticity, and their propensity to regenerate an organism from a single cell. Methods and differentiation protocols used in plant physiology and biology usually involve macroscopic vessels and containers that make difficult, for example, to follow the fate of the same protoplast all along its full development cycle, but also to perform continuous studies of the influence of various gradients in this context. These limits have hampered the precise study of regeneration processes. Results Herein, we present the design of a comprehensive, physiologically relevant, easy-to-use and low-cost microfluidic and microscopic setup for the monitoring of Physcomitrella patens ( P. patens ) growth and development on a long-term basis. The experimental solution we developed is made of two parts (i) a microfluidic chip composed of a single layer of about a hundred flow-through microfluidic traps for the immobilization of protoplasts, and (ii) a low-cost, light-controlled, custom-made microscope allowing the continuous recording of the moss development in physiological conditions. We validated the experimental setup with three proofs of concepts: (i) the kinetic monitoring of first division steps and cell wall regeneration, (ii) the influence of the photoperiod on growth of the protonemata, and (iii) finally the induction of leafy buds using a phytohormone, cytokinin. Conclusions We developed the design of a comprehensive, physiologically relevant, easy-to-use and low-cost experimental setup for the study of P. patens development in a microfluidic environment. This setup allows imaging of P. patens development at high resolution and over long time periods. Electronic supplementary material The online version of this article (10.1186/s13007-019-0459-z) contains supplementary material, which is available to authorized users.

Imaging the living plant cell: From probes to quantification

“…Contrary to the previous system, nutrient medium can circulate between chambers, allowing long-term kinetic experiments to be performed. Using such a system combined with a microscope set-up, a recent study investigated the influence of photoperiod on the growth of the moss Physcomitrella patens (Sakai et al, 2019). By adding hormones to the circulating medium, the authors also observed the induction of leafy buds (Figure 4D) (Sakai et al, 2019).…”

“…Using such a system combined with a microscope set-up, a recent study investigated the influence of photoperiod on the growth of the moss Physcomitrella patens (Sakai et al, 2019). By adding hormones to the circulating medium, the authors also observed the induction of leafy buds (Figure 4D) (Sakai et al, 2019). In another study, a microfluidic platform was designed with microelectrodes, coupled with electrical impedance spectroscopy, to study primary cell wall regeneration at the single-cell level (Chen et al, 2020) (Figure 4E).…”

Current State and Future Directions of Genetics and Genomics of Endophytic Fungi for Bioprospecting Efforts

“…The ease of use of these ready-to-use ORNL devices might make them widely applicable to study endophytic fungi and their BGCs in the presence of their host plants and allow for the tracking of their dynamics at high spatial and temporal resolution. Given that plant development from a single protoplast has been studied in microfluidic devices before (Sakai et al, 2019 ), we could even imagine using a similar setup to study the reciprocal influence of microbiota and plants with such devices. This would be particularly interesting for aerial parts of plants rather than roots and might allow for downstream metabolomics analysis and secondary metabolite discovery.…”

Protoplast: A Valuable Toolbox to Investigate Plant Stress Perception and Response

“…For instance, protoplasts have been employed to assess the efficiency of CRISPR-associated protein 9 (Cas9) mutagenesis, bypassing or preceding stable transformation which can be time-consuming ( Lin et al, 2018 ; Hahn et al, 2020 ; Guyon-Debast et al, 2021 ; Nicolia et al, 2021 ). Another example is the adaptation of efficient and low-cost microfluidic techniques to perform spatiotemporal studies of plant protoplasts physiology during their development ( Sakai et al, 2019 ) and to apprehend the electrical resistance of CW-regenerated protoplasts ( Chen, 2020 ). Similarly, the usefulness of protoplasts for high-throughput RNA sequencing has also been put forward due to its many advantages over traditional RNA-seq.…”