Optimizing Protocols for Arabidopsis Shoot and Root Protoplast Cultivation
Open Access
- 15 February 2021
- Vol. 10 (2), 375
- https://doi.org/10.3390/plants10020375
Abstract
Procedures for the direct regeneration of entire plants from a shoot and root protoplasts of Arabidopsis thaliana have been optimized. The culture media for protoplast donor-plant cultivation and protoplast culture have been adjusted for optimal plant growth, plating efficiency, and promotion of shoot regeneration. Protocols have been established for the detection of all three steps in plant regeneration: (i) chromatin relaxation and activation of auxin biosynthesis, (ii) cell cycle progression, and (iii) conversion of cell-cycle active cells to totipotent ones. The competence for cell division was detected by DNA replication events and required high cell density and high concentrations of the auxinic compound 2,4-D. Cell cycle activity and globular structure formation, with subsequent shoot induction, were detected microscopically and by labeling with fluorescent dye Rhodamine123. The qPCR results demonstrated significantly upregulated expression of the genes responsible for nuclear reorganization, auxin responses, and auxin biosynthesis during the early stage of cell reprogramming. We further optimized cell reprogramming with this protocol by applying glutathione (GSH), which increases the sensitivity of isolated mesophyll protoplasts to cell cycle activation by auxin. The developed protocol allows us to investigate the molecular mechanism of the de-differentiation of somatic plant cells.Keywords
This publication has 50 references indexed in Scilit:
- Characterization of the Early Events Leading to Totipotency in an Arabidopsis Protoplast Liquid Culture by Temporal Transcript ProfilingTHE PLANT CELL ONLINE, 2013
- The main auxin biosynthesis pathway in ArabidopsisProceedings of the National Academy of Sciences of the United States of America, 2011
- Metalloproteomes: A Bioinformatic ApproachAccounts of Chemical Research, 2009
- Auxin Synthesized by the YUCCA Flavin Monooxygenases Is Essential for Embryogenesis and Leaf Formation inArabidopsisTHE PLANT CELL ONLINE, 2007
- The Balance between Cell Division and Endoreplication Depends on E2FC-DPB, Transcription Factors Regulated by the Ubiquitin-SCFSKP2A Pathway inArabidopsisTHE PLANT CELL ONLINE, 2006
- Complementary interactions between oxidative stress and auxins control plant growth responses at plant, organ, and cellular levelJournal of Experimental Botany, 2005
- Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT MethodMethods, 2001
- The culture response of Arabidopsis thaliana protoplasts is determined by the growth conditions of donor plantsThe Plant Journal, 1992
- Arabidopsis thaliana as a model system in somatic cell genetics I. Cell and tissue culturePlant Science Letters, 1975
- A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue CulturesPhysiologia Plantarum, 1962