Isolation, Purification, and Application of Protoplasts and Transient Expression Systems in Plants

Abstract

Protoplasts, derived from plant cells, exhibit remarkable totipotency and hold significant value across a wide spectrum of biological and biotechnological applications. These versatile applications encompass protein subcellular localization and interaction analysis, gene expression regulation, functional characterization, gene editing techniques, and single-cell sequencing. Protoplasts’ usability stems from their inherent accessibility and their ability to efficiently incorporate exogenous genes. In this review, we provide a comprehensive overview, including details on isolation procedures and influencing factors, purification and viability assessment methodologies, and the utilization of the protoplast transient expression system. The aim is to provide a comprehensive overview of current applications and offer valuable insights into protoplast isolation and the establishment of transient expression systems in a diverse range of plant species, thereby serving as a valuable resource for the plant science community.

Response surface methodology mediated optimization of phytosulfokine and plant growth regulators for enhanced protoplast division, callus induction, and somatic embryogenesis in Angelica Gigas Nakai

“…Protoplast technology has been recently utilized for genetic engineering and genome editing in plants. Genome-edited single-cell clones have been produced as a result of technologies such as transiently expressing genes in plant protoplasts and genome-editing protoplasts utilizing the CRISPR/Cas9 system, which has resulted in the regeneration of entire plants [ 5 , 34 ]. To achieve all these processes protoplast-to-plant regeneration should be thoroughly established.…”

“…To achieve all these processes protoplast-to-plant regeneration should be thoroughly established. Even though protoplast regeneration methods have been developed in several plant species, plant regeneration is still a bottleneck in several plant species [ 1 , 5 ].…”

“…The TAL method was developed by Golds et al [ 43 ] and alginate-embedding methods have been widely used for protoplast culture, callus induction, and plant regeneration in many plant species including Arabidopsis thaliana , Beta vulgaris , Daucus carota , Nigella damascena , Platycodon grandifloras , and Nicotiana tobacco [ 30 , 44 – 47 ]. Alginate-based hydrogels have been reported to immobilize the protoplast efficiently and facilitate the immobilized protoplasts to regain the cell walls, cell division, and callus formation [ 1 , 5 ].…”

“…Protoplast fusion opens up new avenues in overcoming the barriers of sexual reproduction and increasing the variability among cultivated plants by nuclear and cytoplasmic gene transfer [ 3 , 4 ]. In addition, insertion, deletion of genes, and gene editing are possible through the transient transformation of protoplasts and by the use of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technologies [ 5 ]. Furthermore, protoplasts are valuable tools in the areas of molecular biology, genetics, and physiological studies [ 1 , 2 ].…”

An efficient method for the isolation and transfection of Pea (Pisum sativum) mesophyll protoplasts

“…Without their cell walls, plant cells become vulnerable for mechanical damage that can occur during the subsequent centrifugation and resuspension steps (21). Moreover, it is important to identify solutions that can maintain the osmotic balance of cells and prevent them from collapsing (20).…”