Optimization of protoplast regeneration in the model plant Arabidopsis thaliana

• Published in: Plant methods 2021-02, Vol.17 (1), p.21-21, Article 21
• Main Authors: Jeong, Yeong Yeop, Lee, Hun-Young, Kim, Suk Weon, Noh, Yoo-Sun, Seo, Pil Joon
• Format: Article
• Language: English
• Subjects: Alginates; Alginic acid; Arabidopsis; Arabidopsis thaliana; Callus; callus formation; Cell culture; Cell cycle; Cell division; Cell growth; Cell walls; CRISPR; De novo organogenesis; Ecotypes; Enzymes; Flowers & plants; Genetic engineering; genome; Genome editing; Genomes; Hydrogels; Optimization; Organogenesis; Physiological aspects; Plant cells and tissues; Plant regeneration; Pluripotency; Polyethylene glycol; Protoplast; Protoplasts; Regeneration; Regeneration (Botany); Seedlings; Sodium; Sucrose; Tissue culture; Tissue engineering; tissue repair
• ISSN: 1746-4811; 1746-4811
• DOI: 10.1186/s13007-021-00720-x

Plants have a remarkable reprogramming potential, which facilitates plant regeneration, especially from a single cell. Protoplasts have the ability to form a cell wall and undergo cell division, allowing whole plant regeneration. 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. To conduct these studies, a reproducible and efficient protoplast regeneration method using model plants is necessary. Here, we optimized cell and tissue culture methods for improving protoplast regeneration efficiency in Arabidopsis thaliana. 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). Among these ecotypes, Ws-2 showed the highest potential for protoplast regeneration. A modified thin alginate layer was applied to the protoplast culture at an optimal density of 1 × 10  protoplasts/mL. Following callus formation and de novo shoot regeneration, the regenerated inflorescence stems were used for de novo root organogenesis. The entire protoplast regeneration process was completed within 15 weeks. The in vitro regenerated plants were fertile and produced morphologically normal progenies. The cell and tissue culture system optimized in this study for protoplast regeneration is efficient and reproducible. This method of Arabidopsis protoplast regeneration can be used for fundamental studies on pluripotency establishment and de novo tissue regeneration.