Optimization of protoplast regeneration in the model plant Arabidopsis thaliana

Abstract

Background 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. Results 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 × 106 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. Conclusion 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.

Embryogenic Stem Cell Identity after Protoplast Isolation from Daucus carota and Recovery of Regeneration Ability through Protoplast Culture

“…The protoplast solution isolated from EC and NEC was embedded in culture using a thin alginate layer (TAL) system, according to Jeong et al [ 47 ]. We mixed 1 mL of protoplast solution (2.0 × 10 6 /mL) with an equal volume of sodium alginate solution ( Table S1 ).…”

All‐in‐one: a robust fluorescent fusion protein vector toolbox for protein localization and BiFC analyses in plants

“…The Vn‐ and Vc‐fused BiFC vector modules enable the N‐ and C‐terminal fusion of two genes of interest to each tag, which could be used for (i) PPI analysis of two proteins of interest, (ii) colocalization analysis of two interacting proteins with organelle markers or (iii) colocalization analysis of two interacting proteins with another protein. The most commonly used approaches for introducing these plasmids into plant cells are PEG/Ca‐mediated transient transformation of protoplasts, biolistic‐mediated transient/stable transformation and Agrobacterium ‐mediated transient/stable transformation (Chen et al ., 2006 ; Jeong et al ., 2021 ; Ramkumar et al ., 2020 ). Of these techniques, protoplast transformation is a rapid and efficient method that has become a widely used tool for plant biology studies (He et al ., 2016 ; Yoo et al ., 2007 ).…”

Protoplast Regeneration and Its Use in New Plant Breeding Technologies

“…When comparing four different Arabidopsis ( Arabidopsis thaliana ) ecotypes (Col-0, Ws-2, No-0, and HR-10), all gave a similar number of protoplasts with an optimized digestion, but differed significantly when comparing optimal protoplast division media, callus induction media, and shoot induction media ( Jeong et al, 2021 ). Ws-2 showed the highest regeneration efficiency, whereas the Col-0, No-0, and HR-10 had relatively ineffective regeneration rates, regardless of efforts to vary the composition of media and tissue culture methods.…”

“…Only 1% sucrose and 2% glucose led to microcallus formation, with 1% sucrose more rapidly producing larger microcalli. For Arabidopsis seedling protoplast culture, three different variations of supplements with B5 medium and vitamins were tested for protoplast proliferation ( Jeong et al, 2021 ). Myo-inositol as the primary carbon source along with sucrose resulted in the highest proliferation rate across four the different Arabidopsis ecotypes.…”