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.
Highlights
Arabidopsis thaliana represents the best-studied model among higher plants
The protoplast plating efficiency is strongly dependent on the donor plant's growing conditions, the medium composition and light conditions [10]. This point is especially true for Arabidopsis, which is a small plant with a short vegetative period that is accompanied by the rapid exit of leaf cells from the cell cycle [11], as well as rapid DNA reduplication that is promoted by high nitrate concentrations [12]
We tested more than 100 different nutrient combinations for Arabidopsis and determined that Optimized Arabidopsis medium 1 (TK1) [9] with an NPK ratio of 5:1:3, which is close to the classical Hewitt nutrient solution ratio [13,14] is optimal for in vitro plant culture
Summary
Arabidopsis thaliana represents the best-studied model among higher plants. Numerous investigations have been performed to characterize the fundamental mechanisms underlying totipotency, pluripotency, and nuclear reprogramming, including epigenetic regulation The heterogeneity of the cells in Arabidopsis tissues complicates any attempts to track the developmental lineage and limits the application of state-of-the-art gene expression methods, such as microarrays and proteomics, which require a population of homogeneous cells. These limitations have been described recently and have been extended to include the additional complexity presented by individual organs that contain cells with different responses to stimuli and different regenerative potentials [2]. Only a restricted number of cells in the plant body maintain their full regenerative potential, while other cells very rapidly lose this capacity because of their rapid differentiation, polyploidization, and an inability to enter into the cell cycle
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