Immobilization Of Protoplasts Research Articles

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

BackgroundPlant 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.ResultsHerein, 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.ConclusionsWe 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.

A simple and effective method to encapsulate tobacco mesophyll protoplasts to maintain cell viability

Protoplasts have been widely used for genetic transformation, cell fusion, and somatic mutation due to the absence of a cell wall. However, without the protection of a cell wall, protoplasts are easy to rupture and aggregate during washing, collecting, and gene transfection. In this work, we propose a simple and effective silica/alginate two-step method to immobilize protoplasts with advantages in experimental manipulation and microscopic imaging, as well as in potentially studying cell biological processes such as secretion and metabolism. The proposed two-step immobilization method adopts Transwell with clear tissue culture-treated membrane to support protoplasts in the form of uniform thin layer, which has three unique properties.•The tissue culture-treated membrane has a good affinity for the plant cell; thus, protoplasts can spread evenly and form a very thin layer.•There are more choices for membrane pore size, depending on the application.•It is very convenient to change or collect the solution without mechanically disturbing the protoplasts. This simple and effective silica sol–gel/alginate two-step immobilization of protoplasts in Transwell has great potential for applications in genetic transformation, metabolite production, and migration assays.

Progress towards sugar beet improvement through somatic hybridization I. Inactivation of nuclei and cytoplasm in donor and recipient protoplasts

The isolation and culture of suspension-derived protoplasts from two sugar beet (<em>Beta vulgaris</em> L.) genotypes are described. Immobilization of protoplasts in agarose resulted in high frequency divisions and microcallus regeneration, with plating efficiency (PE) being clearly genotype-dependent. In further studies towards asymmetric fusion experiments, the effect of different doses of ultraviolet radiation (UV) and iodoacetic acid (IA) on protoplast physiology was assessed. Viability of both treated (UV, IA) and untreated protoplasts (control) was determined by FDA staining, and the biological effect was evaluated by testing the ability of protoplasts to divide and to form calli. The results are discussed in terms of the applicability of the methods for the production of asymmetric protoplasts suitable for somatic hybridization within the genus <em>Beta</em>.

Growth and alkaloid synthesis in cell lines ofCatharanthus roseus obtained through immobilization of cells and protoplasts

Cells and protoplasts ofCatharanthus roseus were immobilized with sodium alginate, agar and agarose. Cells proliferating from the matrix were established separately in liquid suspension and 5 cell lines were isolated which showed differences in their growth and alkaloid synthetic pattern. Cell lines obtained through immobilization of protoplasts yielded higher levels of alkaloids.

New form of lignolytically active mycelium generated by immobilization of protoplasts isolated from the white rot fungi Heterobasidion annosum and Polyporus pinsitus

Immobilized mycelia regenerated from immobilized protoplasts isolated from lignin-degrading Basiodiomycetes have been shown to be able to decompose specifically 14C-labelled dehydropolymers of coniferylalcohol (DHP-lignin) and monomeric lignin-related compounds more intensively than native mycelium, by decarboxylation, demethylation, ring and side chain cleavage. Protoplasts of two white rot fungi were immobilized by entrapment in Na- alginate gel and remained intact after the immobilization procedure. Within the first 3 days of incubation in culture medium, regeneration of hyphal cells occurred. Since hyphal cells regenerated from protoplasts within gel beads were hindered from stretching by the matrix, the microbial immobilized cells differed from native mycelium in terms of their morphology. The time course and extent of lignin degradation by native mycelium and regenerated mycelium of the examined white rot fungi also differed, a sign that there may also be differences between them in terms of the physiology of lignin degradation.

Induction of efficient cell division in alfalfa protoplasts

Alfalfa (Medicago sativa L.) protoplasts derived from cell suspension cultures divided inefficiently in liquid culture. The onset of cell division activity occurred synchronously among the protoplasts; however, many were blocked at cytokinesis and therefore did not complete first division. Very few of the cells that began to divide continued to do so. Immobilization of protoplasts in agarose after 1 to 4 days in liquid culture overcame this inhibition of division. Continuous growth in agarose was restricted and therefore microcolonies were transferred to agar medium to complete callus development. Plating efficiencies of 2-10% were achieved within 30 days of protoplast isolation. The agarose treatment was responsible for a 5- to 30-fold improvement in plating efficiency.

Immobilization of protoplasts by anchoring to microcarriers

Datura innoxia cell suspension-derived protoplasts were anchored to Cytodex 1 microcarriers pre-swollen in buffered concanavalin A. As many as 34 protoplasts were estimated to attach per microcarrier, in comparison to a potential 47 as determined from a model based on random anchorage. Fluorescein diacetate was used as localizing agent as well as to assess viability. When included in the swelling medium fluorescence was observed almost instantaneously, first in the protoplast at its interface with the microcarrier, and later throughout the cytoplasm. However, the dye was not conjugated with the lectin, and leakage eventually resulted in fluorescence also of non-anchored protoplasts. Fluorescein-labelled concanavalin A on the other hand permitted detection of microcarriers but not of anchored protoplasts, suggesting the use of differentially fluorescing microcarriers, as an aid in identification of fusion partners.