Sep 16, 2016
- Steven Burgess1
- 1University of Cambridge
- OpenPlant Project
External link: https://doi.org/10.1371/journal.pone.0196810
Protocol Citation: Steven Burgess 2016. Plant cell protoplast isolation. protocols.io https://dx.doi.org/10.17504/protocols.io.ftnbnme
Manuscript citation:
Yu Z, Boehm CR, Hibberd JM, Abell C, Haseloff J, Burgess SJ, Reyna-Llorens I (2018) Droplet-based microfluidic analysis and screening of single plant cells. PLoS ONE 13(5): e0196810. doi: 10.1371/journal.pone.0196810
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: September 14, 2016
Last Modified: March 21, 2018
Protocol Integer ID: 3662
Abstract
A protocol for isolation of protoplast from plant cells adapted from Yoo et al. 2007 (http://www.nature.com/nprot/journal/v2/n7/full/nprot.2007.199.htm). Procedure was optimized for use on Arabidopsis thaliana tissue.
Safety warnings
This protocol worked best for dicot species, yields from monocot leaves were low.
Grow plants of Arabidopsis thaliana (ecotype Col0) on a 3:1 (v/v) compost to vermiculite mixture at 20oC, under short day conditions (12h light:12h dark) and low light intensities (50-100µE m-2 s-1).
Prepare fresh enzyme solution and transfer to glass petridish.
Protocol
NAME
Protoplast Isolation - Enzyme BufferCREATED BY
Steven J Burgess
500mM MES, pH 5.6
1 mL
Note
Final concentration is 1.5% (w/v)
Note
Final concentration is 10mM. Yoo et al. 2007 mention that MES is preheated to 70oC for 3-5 minutes prior to addition of the enzyme powder.
Mannitol
5 g
Note
Final Concentration is 0.3% (w/v)
Note
Final concentration is 1.5% (w/v)
Note
Final concentration is 0.6M
1M Potassium Chloride
KCl
1 mL
Note
Final Concentration is 0.3% (w/v)
Note
Final concentration is 1.5% (w/v)
Note
Final concentration is 20μM
Note
Final concentration is 0.6M
Note
Final concentration is 10mM. Yoo et al. 2007 mention that MES is preheated to 70oC for 3-5 minutes prior to addition of the enzyme powder.
Add dH2O up to 50mL
Note
Final Concentration is 0.3% (w/v)
Note
Final concentration is 1.5% (w/v)
Note
Final concentration is 20μM
Note
Final concentration is 0.6M
Note
Final concentration is 10mM. Yoo et al. 2007 mention that MES is preheated to 70oC for 3-5 minutes prior to addition of the enzyme powder.
Cellulase R10
Note
Final Concentration is 0.3% (w/v)
Note
Final concentration is 1.5% (w/v)
Note
Final concentration is 20μM
Macerozyme R10
Note
Final Concentration is 0.3% (w/v)
Heat the enzyme solution at 55oC for 10 min, then allow to cool to room temperature.
1M Calcium Chloride
CaCl2
50 µL
Filter final solution through a 0.45-μm syringe filter.
Remove 20-40 leaves from 4 week old Arabidopsis thaliana rosettes.
Pile up 5-10 leaves at a time, cut into thin (~2mm) strips across the leaf using a sharp scapel blade dipped into ethanol. Place strips into enzyme solution. Repeat until all leaves have been cut.
Vacuum infiltrate the enzyme solution.
00:30:00
Leave cells in the petri dish at 20oC without shaking.
04:00:00
Twice filter cells and enzyme solution through a 125μm nylon mesh to remove residual debris into a pointed bottom, glass test tube.
Leave cells to settle to the bottom of the tube.
00:30:00
Carefully aspirate out the liquid solution leaving the cell pellet intact.
Add Incubation Buffer
1 mL
Protocol
NAME
Protoplast Isolation - Incubation BufferCREATED BY
Steven J Burgess
Mannitol
5 g
0.5M MES, pH 5.6
1 mL
Note
Final [10mM]
Potassium chloride
1M KCl
200 µL
Note
Final [4mM]
Gently resuspend protoplast in incubation solution by light swirling of the test tube.
Note
Note: You cannot view protoplasts on a flat glass slide with a coverslip as they will burst.
Note
Protoplasts are extremely fragile, it is best to avoid pipetting up and down or shaking vigorously as the shearing forces can cause cells to rupture.
Check protoplast integrity and determine yield using a disposable hemocytomer.
Note
Note: You cannot view protoplasts on a flat glass slide with a coverslip as they will burst.
Note
Diltute cells as necessary with extra incubation buffer.