Optimizing Protocols for Arabidopsis Shoot and Root Protoplast Cultivation

Abstract

Procedures for the direct regeneration of entire plants from a shoot and root protoplasts of Arabidopsis thaliana have been optimized. The culture media for protoplast donor-plant cultivation and protoplast culture have been adjusted for optimal plant growth, plating efficiency, and promotion of shoot regeneration. Protocols have been established for the detection of all three steps in plant regeneration: (i) chromatin relaxation and activation of auxin biosynthesis, (ii) cell cycle progression, and (iii) conversion of cell-cycle active cells to totipotent ones. The competence for cell division was detected by DNA replication events and required high cell density and high concentrations of the auxinic compound 2,4-D. Cell cycle activity and globular structure formation, with subsequent shoot induction, were detected microscopically and by labeling with fluorescent dye Rhodamine123. The qPCR results demonstrated significantly upregulated expression of the genes responsible for nuclear reorganization, auxin responses, and auxin biosynthesis during the early stage of cell reprogramming. We further optimized cell reprogramming with this protocol by applying glutathione (GSH), which increases the sensitivity of isolated mesophyll protoplasts to cell cycle activation by auxin. The developed protocol allows us to investigate the molecular mechanism of the de-differentiation of somatic plant cells.

An Efficient and Universal Protoplast Isolation Protocol Suitable for Transient Gene Expression Analysis and Single-Cell RNA Sequencing

“…As shown in Figure 2 , we found that ~89% of the cells in the protoplast suspension are viable, as indicated by their round shape with strong fluorescent signals ( Supplemental Figure S4 ). This result was further substantiated by the time-lapse Rhodamine 123 staining [ 47 ] with most of the cells exhibit fluorescent signals in the corresponding time point ( Supplemental Figures S5 and S6 ).…”

Establishing a reproducible approach for the controllable deposition and maintenance of plants cells with 3D bioprinting

“…Previous studies have shown that cellular regeneration by callus formation from root explants initiates with divisions of pericycle cells, rather than dedifferentiation and cell cycle re-entry of root cells (17). To our knowledge, cell cycle re-entry of protoplasts derived from Arabidopsis roots has not yet been achieved (11,17). To understand whether bioprinted meristematic and differentiated root cells enter the cell cycle, we evaluated the bioprinted cells for divisions and microcallus formation.…”

“…Interestingly, the formed microcalli expressed the endodermal marker SCR. Previous studies showed that callus formation from roots explants was initiated by pericycle cells differentiating into cells resembling root meristems (11,17). However, the expression of SCR in the bioprinted microcalli indicates that in addition to pericycle-derived microcallus formation, another reprogramming mechanism might trigger microcallus formation.…”

“…To achieve long-term cell viability, we optimized tissue-specific bioinks and established a reproducible experimental pipeline that consists of bioprinting protoplasts, and maintaining and imaging the bioprinted constructs over time (Fig 1A). In this pipeline, we focused on Arabidopsis thaliana roots, a tissue of which the maintenance outside its context is known to be technically challenging (11). Additionally, to further gain insights into whether different tissue types would be more prone to cellular reprogramming, we isolated protoplasts from both meristematic and differentiated root tissue and performed comparative analyses.…”

Adaptive diversification in the cellular circadian behavior of Arabidopsis leaf- and root-derived cells

“…As previously reported 27–30 , we prepared protoplasts from the leaves and roots of an Arabidopsis CCA1::LUC transgenic plant. Protoplasts of leaves are mainly mesophyll cells 27 and those of roots are presumedly a heterogeneous population with different cell types 31,32 . Cells derived from the protoplasts of leaves and roots are hereafter referred to as leaf-derived cells and root-derived cells, respectively.…”