Protoplasm isolation is a recurrent technique that is needed for the transfection of plant tissue culture and the evaluation of transiently expressed genetic vectors. This is a widely used technique in many plant development labs. However further adaptation might require adjustments on either more recalcitrant salt and buffer concentrations or longer exposure to enzymatic hydrolysis. In some cases, cellulase 10 is indicated for woody tissues with longer incubation times. Here we present a protocol that is adapted from Zhang et al. Plant Methods 2011, 7:30 http://www.plantmethods.com/content/7/1/30, usually intended for monocots (e.g. grasses). However, this protocol can be adjusted for other types of plants. The complementary video helps to visualize the most critical steps. https://www.youtube.com/watch?v=LjqmE3mnrQU
Rice Protoplast isolation (can be adapted to other monocots)
Zhang et al. Plant Methods 2011, 7:30 http://www.plantmethods.com/content/7/1/30
MATERIAL & SOLUTIONS:
Sodium hypochlorite 2.5%
1/2 MS medium
Enzyme solution: 1.5% Cellulase RS, 0.75% Macerozyme R-10, 0.6 M mannitol, 10 mM MES at pH 5.7, 10 mM CaCl2 and 0.1% BSA
W5 solution: 154 mM NaCl, 125 mM CaCl2, 5 mM KCl and 2 mM MES at pH 5.7
MMG solution: 0.4 M mannitol, 15 mM MgCl2 and 4 mM MES at pH 5.7
Sharp sterile razor or scissors.
Orbital shaker or rotator
40 μm nylon meshes (for 50ml falcon tubes)
Micro pipettes (1ml-5ml)
Culture vessels (e.g. Magenta boxes), petri dishes, falcon tubes
Preparation of material and seed germination:
-Sterilize de-hulled seeds with 75% EtOH for1min
-Discard used EtOH
-Sterilize de-hulled seeds with 2.5% sodium hypochlorite for 20min
-Discard Sodium hypochlorite solution
-Wash seeds 5x with sterilized DDH2O
-Incubate with 1/2 MS medium with a photoperiod of 12 h light (about 150 μmol m-2 s-1) and 12 h dark at 26°C for 7-10 days.
-Green tissues were extracted from the stem and sheath of 40-60 rice seedlings. A bundle of rice plants (about 30 seedlings) was propulsively cut into approximately 0.5 mm strips with sharp razor blades.
-Transfer strips into 0.6 M mannitol for 10 min in the dark
- Incubate strips in enzyme solution (1.5% Cellulase RS, 0.75% Macerozyme R-10, 0.6 M man- nitol, 10 mM MES at pH 5.7, 10 mM CaCl2 and 0.1% BSA) for 4-5 h in the dark with gentle shaking (60-80 rpm).
-Add an equal volume of W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl and 2 mM MES at pH 5.7) and vigorous shaking for 10 sec
-Filter protoplasms through 40 μm nylon meshes
-Wash 3-5x using W5 solution
Recover protoplasm by centrifugation at 1,500 rpm for 3 min with a swinging bucket (fixed angle rotor can work too, reduce speed for 1,000 rpm for 5 mins)
Wash again with W5 solution
pellets were then resuspended in MMG solution (0.4 M mannitol, 15 mM MgCl2 and 4 mM MES at pH 5.7)
-Determine cells concentration using a hematocytometer
Further discussion and adaptations to the Protoplasma isolation can be found here: https://molbio.mgh.harvard.edu/sheenweb/faq.html
All manipulations above were performed at room temperature.
Where to finds some of the tools enlisted here
40 μm nylon meshes
Article TitleA highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes
Background: Plant protoplasts, a proven physiological and versatile cell system, are widely used in high-throughput analysis and functional characterization of genes. Green protoplasts have been successfully used in investigations of plant signal transduction pathways related to hormones, metabolites, and environmental challenges. In rice, protoplasts are commonly prepared from suspension-cultured cells or etiolated seedlings, but only a few studies have explored the use of protoplasts from rice green tissue.
Results: Here, we report a simplified method for isolating protoplasts from normally cultivated young rice green tissue without the need for unnecessary chemicals and a vacuum device. Transfections of the generated protoplasts with plasmids of a wide range of sizes (4.5-13 kb) and co-transfections with multiple plasmids achieved impressively high efficiencies and allowed evaluations by 1) protein immunoblotting analysis, 2) subcellular localization assays, and 3) protein-protein interaction analysis by bimolecular fluorescence complementation (BiFC) and firefly luciferase complementation (FLC). Importantly, the rice green tissue protoplasts were photosynthetically active and sensitive to the retrograde plastid signaling inducer norflurazon (NF). Transient expression of the GFP- tagged light-related transcription factor OsGLK1 markedly upregulated transcript levels of the endogenous photosynthetic genes OsLhcb1, OsLhcp, GADPH, and RbcS, which were reduced to some extent by NF treatment in the rice green tissue protoplasts.
Conclusions: We show here a simplified and highly efficient transient gene expression system using photosynthetically active rice green tissue protoplasts and its broad applications in protein immunoblot, localization, and protein-protein interaction assays. These rice green tissue protoplasts will be particularly useful in studies of light/chloroplast-related processes.