3.2 A. thaliana
Seedlings
3.2.1 Arabidopsis Seed
Sterilization
All steps requiring opening of a tube need to be performed under a
sterile hood.
1. Prepare around 50 μL of A. thaliana Col-0 seeds in a 1.5 mL
microtube (see Note 9).
2. Add 1 mL of seed sterilization solution. Mix to resuspend the
seeds.
3. Gently mix for 15 min on a tube rotator.
4. Centrifuge at 3000 Â g for 10 s to pellet the seeds.
5. Carefully remove the supernatant.
6. Add 1 mL of 100% ethanol and mix to resuspend the seeds.
7. Gently mix on a tube rotator for 2 min.
8. Centrifuge at 3000 Â g for 10 s to pellet the seeds.
9. Remove as much supernatant as possible.
10. Repeat steps 6–9 one more time.
11. Gently hit the tube with a finger to spread seeds, open the tube,
and allow the seeds to dry for 1 h.
3.2.2 Growing
Arabidopsis Seedlings
1. Heat seedling growth medium in a microwave to melt
it. Under a sterile hood, pour 50 mL of the medium into a
square Petri dish (120 mm  120 mm) and let the dish open
until the medium is solidified.
2. Scatter the sterilized seeds onto the solidified medium.
3. Seal the dish with micropore gas-permeable tape.
4. Allow the seeds to vernalize by keeping the dish at 4
C for
2–5 days.
5. Let seedlings grow for 7 days under long day conditions (8-h
dark at 17
C, 16-h light at 21
C).
3.3 Isolation
of Protoplasts
3.3.1 Preparation
of Digestion Buffer
Digestion buffer must be prepared just before use. The following
protocol is given for 50 mL digestion buffer.
1. Preheat a water bath to 55
C.
2. Weigh 0.75 g of cellulase and 0.2 g of macerozyme and transfer
to a 50 mL tube.
3. Add 40 mL of 0.5 M mannitol, pH 5.7, 2.5 mL of 0.4 M MES,
and 500 μL of 2 M KCl. Vortex and heat at 55
C until enzymes
are completely dissolved.
4. Add 500 μL of 1 M CaCl 2 and 500 μL of acetylated BSA.
Adjust volume to 50 mL with water and gently mix.
3.3.2 Removal of Cell
Walls from Cultured Cells
25 mL of digestion buffer per 10 mL of cell culture is required.
420
Ste ´ phanie Lalande et al.
Seedlings
3.2.1 Arabidopsis Seed
Sterilization
All steps requiring opening of a tube need to be performed under a
sterile hood.
1. Prepare around 50 μL of A. thaliana Col-0 seeds in a 1.5 mL
microtube (see Note 9).
2. Add 1 mL of seed sterilization solution. Mix to resuspend the
seeds.
3. Gently mix for 15 min on a tube rotator.
4. Centrifuge at 3000 Â g for 10 s to pellet the seeds.
5. Carefully remove the supernatant.
6. Add 1 mL of 100% ethanol and mix to resuspend the seeds.
7. Gently mix on a tube rotator for 2 min.
8. Centrifuge at 3000 Â g for 10 s to pellet the seeds.
9. Remove as much supernatant as possible.
10. Repeat steps 6–9 one more time.
11. Gently hit the tube with a finger to spread seeds, open the tube,
and allow the seeds to dry for 1 h.
3.2.2 Growing
Arabidopsis Seedlings
1. Heat seedling growth medium in a microwave to melt
it. Under a sterile hood, pour 50 mL of the medium into a
square Petri dish (120 mm  120 mm) and let the dish open
until the medium is solidified.
2. Scatter the sterilized seeds onto the solidified medium.
3. Seal the dish with micropore gas-permeable tape.
4. Allow the seeds to vernalize by keeping the dish at 4
C for
2–5 days.
5. Let seedlings grow for 7 days under long day conditions (8-h
dark at 17
C, 16-h light at 21
C).
3.3 Isolation
of Protoplasts
3.3.1 Preparation
of Digestion Buffer
Digestion buffer must be prepared just before use. The following
protocol is given for 50 mL digestion buffer.
1. Preheat a water bath to 55
C.
2. Weigh 0.75 g of cellulase and 0.2 g of macerozyme and transfer
to a 50 mL tube.
3. Add 40 mL of 0.5 M mannitol, pH 5.7, 2.5 mL of 0.4 M MES,
and 500 μL of 2 M KCl. Vortex and heat at 55
C until enzymes
are completely dissolved.
4. Add 500 μL of 1 M CaCl 2 and 500 μL of acetylated BSA.
Adjust volume to 50 mL with water and gently mix.
3.3.2 Removal of Cell
Walls from Cultured Cells
25 mL of digestion buffer per 10 mL of cell culture is required.
420
Ste ´ phanie Lalande et al.