![]() Liquid culture of protoplasts is a simple and easy technique used to induce cell division and callus formation, but it has a low efficiency of tissue regeneration, owing to cell aggregation-induced cell death and low cell proliferation activity. Conventional methods of protoplast regeneration involve liquid culture. Protoplast regeneration methods have been developed in several plant species. Understanding the molecular mechanisms underlying protoplast regeneration will further advance plant cell-based biotechnological applications, such as genome editing and somatic cell hybridization. Protoplast regeneration is distinct from tissue explant-derived plant regeneration, and molecular processes involved in cell fate transition during protoplast regeneration are largely unknown. įundamental studies on key processes involved in protoplast regeneration, including cell wall recovery, cell cycle re-entry, callus formation, pluripotency acquisition, and de novo tissue regeneration, are essential. The RNPs have been successfully introduced into various plant species, such as Arabidopsis thaliana, tobacco ( Nicotiana attenuata), lettuce ( Lactuca sativa L.), rice ( Oryza sativa L.), petunia ( Petunia × atkinsiana), and potato ( Solanum tuberosum L.), via polyethylene glycol–calcium (PEG–Ca 2+)-mediated transfection. Moreover, DNA-free genome editing has been developed with the delivery of preassembled Cas9-gRNA ribonucleoproteins (RNPs) into protoplasts derived from somatic tissues. For example, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been transiently expressed in plant protoplasts, and genome-edited protoplasts have been regenerated into individual plants. Given the advantages of improved synchrony initiated from a single cell without sexual reproduction, protoplast regeneration techniques have been widely used for genetic engineering and genome editing in plants. Protoplasts exhibit a remarkable ability to dedifferentiate, and cultured protoplasts have the ability to form cell walls and undergo cell division, allowing whole plant regeneration. Plants have a remarkable reprogramming potential, which facilitates plant regeneration from organs, tissues, and even a single cell. This method of Arabidopsis protoplast regeneration can be used for fundamental studies on pluripotency establishment and de novo tissue regeneration. The cell and tissue culture system optimized in this study for protoplast regeneration is efficient and reproducible. The in vitro regenerated plants were fertile and produced morphologically normal progenies. The entire protoplast regeneration process was completed within 15 weeks. Following callus formation and de novo shoot regeneration, the regenerated inflorescence stems were used for de novo root organogenesis. A modified thin alginate layer was applied to the protoplast culture at an optimal density of 1 × 10 6 protoplasts/mL. Among these ecotypes, Ws-2 showed the highest potential for protoplast regeneration. Protoplasts were isolated from whole seedlings of four different Arabidopsis ecotypes including Columbia (Col-0), Wassilewskija (Ws-2), Nossen (No-0), and HR (HR-10). Here, we optimized cell and tissue culture methods for improving protoplast regeneration efficiency in Arabidopsis thaliana. To conduct these studies, a reproducible and efficient protoplast regeneration method using model plants is necessary. With the growing need for protoplast regeneration in genetic engineering and genome editing, fundamental studies that enhance our understanding of cell cycle re-entry, pluripotency acquisition, and de novo tissue regeneration are essential. Protoplasts have the ability to form a cell wall and undergo cell division, allowing whole plant regeneration. Plants have a remarkable reprogramming potential, which facilitates plant regeneration, especially from a single cell.
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