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

Abstract

The recent advent of single-cell RNA sequencing (scRNA-seq) has enabled access to the developmental landscape of a complex organ by monitoring the differentiation trajectory of every specialized cell type at the single-cell level. A main challenge in this endeavor is dissociating plant cells from the rigid cell walls and some species are recalcitrant to such cellular isolation. Here, we describe the establishment of a simple and efficient protocol for protoplast preparation in Chirita pumila, which includes two consecutive digestion processes with different enzymatic buffers. Using this protocol, we generated viable cell suspensions suitable for an array of expression analyses, including scRNA-seq. The universal application of this protocol was further tested by successfully isolating high-quality protoplasts from multiple organs (petals, fruits, tuberous roots, and gynophores) from representative species on the key branches of the angiosperm lineage. This work provides a robust method in plant science, overcoming barriers to isolating protoplasts in diverse plant species and opens a new avenue to study cell type specification, tissue function, and organ diversification in plants.

Optimization of Isolation and Transformation of Protoplasts from Uncaria rhynchophylla and Its Application to Transient Gene Expression Analysis

“…In maize, pretreatment of the samples with a balanced osmotic buffer prior to digestion significantly increases the efficiency of protoplasts generated [ 12 ]. Therefore, we applied a pretreatment buffer to the U. rhynchophylla samples for 20 min.…”

“…Enzymatic hydrolysis is often used in protoplast preparation, and the enzyme components and conditions used is the most critical factor affecting protoplast separation and activity [ 11 ]. Among many factors, the composition and pH value of the enzyme solution; the temperature, speed, and duration of enzymatic hydrolysis; different enrichment treatments; and the properties of the prefabricated solution all affect the yield and activity of the protoplasts obtained [ 12 ].…”

Research strategies for single‐cell transcriptome analysis in plant leaves

“…The study also highlighted the power of snRNA-seq in identifying unprecedented spatiotemporal gene expression atlas of plant shoot cells (Tian et al, 2020). Similarly, Wang et al (2022) recently developed an efficient protocol, consisting of two digestion processes with several enzymatic buffers, for protoplast preparation in Chirita pumila. This protocol generates viable cell suspensions suitable for scRNA-seq analysis (Wang et al, 2022).…”

“…Similarly, Wang et al (2022) recently developed an efficient protocol, consisting of two digestion processes with several enzymatic buffers, for protoplast preparation in Chirita pumila. This protocol generates viable cell suspensions suitable for scRNA-seq analysis (Wang et al, 2022). This protocol was further applied to isolate high-quality protoplast from several other organs such as fruits and tuberous roots.…”

Advances and applications of single‐cell omics technologies in plant research

“…Accordingly, different cell wall digestion protocols have been developed to release protoplasts from different tissue types in a range of plant species, including Arabidopsis (Birnbaum et al., 2003), tomato ( Solanum lycopersicum ) (Omary et al., 2022), rice ( Oryza sativa ) (Liu et al., 2021), and maize (Bezrutczyk et al., 2021). Efforts have also been made to find more broadly applicable protocols for protoplast isolation (Wang et al., 2022). In comparison with protoplast isolation, nucleus isolation is more robust and can be adapted to a wider range of tissue types and species (Farmer et al., 2021; Marand et al., 2021; Omary et al., 2022; Tian et al., 2020).…”