Protoplast Isolation
Protoplast Isolation - Related Videos
10:12
A Robotic Platform for High-throughput Protoplast Isolation and Transformation
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Cited by 21 •
2016
A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.
11:16
Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana
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Cited by 59 •
2009
This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.
14:20
Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example
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Cited by 20 •
2014
Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.
12:29
mRNA Interactome Capture from Plant Protoplasts
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Cited by 1 •
2017
Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.
13:35
Fluorescence Activated Cell Sorting of Plant Protoplasts
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Cited by 91 •
2010
A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.
FAQs
Cellulase and pectinase remove different structural components of the plant cell wall. Cellulase digests cellulose, while pectinase breaks down pectin, together loosening the wall sufficiently to release the plasma-membrane-bound cell. Their combined action is important because incomplete digestion can limit protoplast recovery, whereas successful wall removal creates a flexible system for subsequent cellular and genetic studies.
Once the cell wall has been removed, the plasma membrane no longer has the same external mechanical support. An osmotic stabilizer helps maintain conditions that reduce the risk of membrane rupture, allowing the released cells to remain living. This protection is central to obtaining usable protoplasts for experiments involving signaling, gene expression, transformation, or regeneration.
The absence of a cell wall gives researchers direct access to cells bounded primarily by the plasma membrane. This makes protoplasts a flexible experimental system for examining membrane function and cellular signaling under controlled laboratory conditions. Their living state also allows investigators to connect membrane-related responses with changes in gene expression and later cellular behavior.
A basic workflow begins with suitable plant, fungal, or algal cells, followed by enzymatic treatment to remove the relevant cell-wall components. An osmotic stabilizer is included to protect the exposed plasma membrane during this process. The resulting living protoplasts can then be directed toward analysis, transient genetic transformation, fusion, or regeneration, depending on the research objective.
Researchers can use isolated protoplasts for transient genetic transformation when they need a flexible living-cell system for examining gene activity or expression. Because the wall has been removed, the cells provide an accessible context for introducing and assessing genetic changes over a limited experimental period. This application connects cell-level manipulation with studies of gene expression and cellular responses.
Protoplasts can be merged through protoplast fusion, creating a basis for somatic hybridization, which combines cellular material without relying solely on conventional reproductive processes. These approaches support plant-breeding research by enabling investigators to explore new cellular combinations and regeneration outcomes. In crop-improvement studies, the technique therefore links controlled cell manipulation with broader efforts to develop useful plant traits.