Rice protoplast isolation and transient expression assay
Songbiao Chen
Plant Growth
Germinate about 100 rice seeds on 1/2 MS media under light for 3 days, then grow seedlings at 26℃ in the dark for about 12~14 days. The mature plant grows in green house can be use for isolation too.
Protoplast Isolation
Using a razor blade, cut the stems and leaves of the seedlings into ~0.5 mm strips.
Put them into a petri dish with 10 ml Enzyme Solution (containing 1.5% cellulase and 0.3% macerozyme).
Apply vacuum for 1 hour for infiltration of the Enzyme Solution.
Incubate for about 4 hours in the dark with gentle shaking (~ 40 rpm) at room temperature.
Remove the Enzyme Solution with a glass pipet.
Add 10ml W5 medium, gently swirl the plate (80 rpm) for 1 hour to release the protoplast.
Transfer the W5 medium containing the protoplasts and filter the protoplasts through a 35 μm nylon mesh.
Transfer the protoplasts into 8 ml glass tube. Centrifuge the tube at 1,500 rpm for 4 minutes to collect the protoplasts. Usually, about 10~14×106 cells can be obtained from 100 seedlings.
DNA Transfection
For tansfection, remove the W5 medium carefully after centrifuge, then re-suspend the protoplasts with appropriate volume of Suspension Medium.
Prepare a 2.0 ml tube containing about 10 μg plasmid DNA (DNA should be resuspended in sterile ddH2O with concentration of about 1μg/1μl).
Add 200 μl (usually 1.5~2.5×106 cells/ml) of suspended protoplasts to the tube containing the plasmid DNA.
Then add 220 ul 40% PEG solution and mix it well immediately by gently shaking, incubate 20 min at room temperature.
Add 1.0 ml W5 medium to the tube to dilute PEG, (If you want to check GFP, use the K3 medium instead of the W5 medium).
Luciferase activity Assay
A LUC assay kit from Promega is used in the assay.
Collect the transfected protoplasts by centrifuging at 2,000 rpm for 5 minutes. Remove the supernatant carefully. Add 200 ul 1×CCLR (Cell Culture Lysis Reagent, from Promega).
Vortex for 30 second, and centrifuge at 2,000 rpm for 5 minutes.
The supernatant cell lysate can be used for both LUC and GUS assay.
Transfer 20 μl cell lysate to the plate and add 100 μl of the Luciferase Assay Reagent to the cell lysate. Then test the LUC with a fluorescence spectrophotometer VICTOR II.
GUS assay
Transfer 150 μl cell lysate to the 1.5 ml tube.
Add 150 ul of 2×GUS assay buffer to 150 μl cell lysate and mix well.
Incubate the assay mix at 37℃ in the dark for about 4-6 hours.
Take 20 μl of assay mix and add 0.2 M Na2CO3 stop the reaction.
Then the assay mix was tested with a fluorescence spectrophotometer VICTOR II.
The GUS assay has been described by Jefferson (Add the cell extract into 100 μl of 1mM MUG, incubate for 60-90min at 37, add 0.9 ml 0.2 M Na2CO3 to stop the reaction, and measure the fluorescence of MU).
GFP Detection
Transfected protoplasts were detected under a Nikon E600 fluorescence microscope. GFP fluorescence was visualized with a filter set consisting of an excitation filter of 450–490 nm, a dichroic mirror of 510 nm, and a barrier filter of 520–560 nm. Images were captured with a SPOT 2 Slider charge-coupled device camera and the associated software (Diagnostics Instruments, Sterling Heights, MI).
Solutions:
1. Enzyme Solution
5% cellulase, 0.3% macerozyme in K3 medium. Filter sterilized, store at -20℃.
2. K3 medium (1 L)
100×B5 Salt mixture / 100 ml1000×B5 Vitamines / 1 ml
200×MES (0.1g/ml) / 5 ml
500×myo-inositol (0.05g/ml) / 2 ml
100×NH3NO3 (25mg/ml) / 10 ml
100×CaCl2 (75mg/ml) / 10 ml
100×Xylose (25mg/ml) / 10 ml
0.4 M Sucrose / 137 g
Adjust pH 5.6-5.8 by 1M KOH, filter-sterilized, store at 4℃.
3. W5 medium (1L)
154 mM NaCl / 9 g125 mM CaCl2 / 18.4 g
5 mM KCl / 0.375 g
2 mM MES / 0.39 g
Adjust pH 5.7 by 1M KOH, filter-sterilized, store at 4℃.
4. Protoplast Suspension Medium
Mannitol / 0.4 MCaCl2 / 20 mM
MES / 5 mM
Adjust pH 5.7 by 1M KOH, filter-sterilized, store at 4℃.
5. PEG Solution
PEG 4000 / 40 %Mannitol / 0.4 M
Ca(NO3)2 / 100 mM
Adjust pH 7.0 by 1M KOH, filter-sterilized, store at -20℃.
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