3.3.3 Removal of Cell
Walls from Seedlings
25 mL of digestion buffer per 200 mg of young seedling leaves is
required.
1. Place 2 mL of digestion buffer into a Petri dish.
2. From a 7-day-old culture of seedlings (Fig. 1b), cut seedlings
with scissors to remove the roots. Weigh 200 mg of the remaining young leaves and transfer them to the Petri dish.
3. Carefully lacerate leaves with a scalpel blade and cover them
with 23 mL of digestion buffer. Lacerated leaves must never be
in contact with air and must not dry.
4. Incubate in a vacuum chamber at 0.01 bar until bubbles
appear. Wait for 1 min to allow buffer infiltration into tissues,
and slowly break vacuum.
5. Cover Petri dish with aluminum foil, and incubate at 26
C
overnight at 50 rpm (orbital shaker).
3.3.4 Purification
of Protoplasts
1. After cell wall digestion (using cells from either cell culture or
seedlings), place a funnel with a single layer of Miracloth on an
Erlenmeyer. Wet the Miracloth with 10 mL of MM buffer.
2. Add 10 mL of MM buffer to the protoplast digestion mixture
(see Note 8). Break cell aggregates by pipetting 2–3 mL of the
mixture with a 10 mL pipette and by creating bubbles by
pipetting air. Pour the cell culture back into Petri dish, and
repeat this operation several times, to allow the release of the
majority of protoplasts. Disappearance of aggregates can be
followed by observing a drop with an inverted microscope.
3. Filter protoplasts through the Miracloth layer and transfer
10 mL aliquots of the filtrate into 14 mL round-bottom
tubes. This step removes the undigested cell walls and tissues.
4. Centrifuge for 3 min at 100 Â g without brake. Remove
supernatant and resuspend the protoplasts by gentle swirling.
5. Add 5 mL of W5 buffer per tube. Gather the content of two
tubes into one. Centrifuge for 1 min at 100 Â g without brake.
6. Remove supernatant and softly resuspend protoplasts.
7. Repeat steps 5 and 6 until all protoplasts are gathered into two
tubes.
8. Gently resuspend protoplasts by inversion of the tubes and add
10 mL of MMG buffer per tube. Centrifuge for 1 min at
100 Â g without brake. Remove supernatant and softly resuspend protoplasts.
9. Add 2 mL of MMG, gather tubes, and incubate for at least
15 min on ice.
10. Add 10 mL of MMG. Centrifuge for 1 min at 100 Â g. Remove
supernatant and softly resuspend protoplasts in 1 or 2 mL
of MMG.
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Ste ´ phanie Lalande et al.
Walls from Seedlings
25 mL of digestion buffer per 200 mg of young seedling leaves is
required.
1. Place 2 mL of digestion buffer into a Petri dish.
2. From a 7-day-old culture of seedlings (Fig. 1b), cut seedlings
with scissors to remove the roots. Weigh 200 mg of the remaining young leaves and transfer them to the Petri dish.
3. Carefully lacerate leaves with a scalpel blade and cover them
with 23 mL of digestion buffer. Lacerated leaves must never be
in contact with air and must not dry.
4. Incubate in a vacuum chamber at 0.01 bar until bubbles
appear. Wait for 1 min to allow buffer infiltration into tissues,
and slowly break vacuum.
5. Cover Petri dish with aluminum foil, and incubate at 26
C
overnight at 50 rpm (orbital shaker).
3.3.4 Purification
of Protoplasts
1. After cell wall digestion (using cells from either cell culture or
seedlings), place a funnel with a single layer of Miracloth on an
Erlenmeyer. Wet the Miracloth with 10 mL of MM buffer.
2. Add 10 mL of MM buffer to the protoplast digestion mixture
(see Note 8). Break cell aggregates by pipetting 2–3 mL of the
mixture with a 10 mL pipette and by creating bubbles by
pipetting air. Pour the cell culture back into Petri dish, and
repeat this operation several times, to allow the release of the
majority of protoplasts. Disappearance of aggregates can be
followed by observing a drop with an inverted microscope.
3. Filter protoplasts through the Miracloth layer and transfer
10 mL aliquots of the filtrate into 14 mL round-bottom
tubes. This step removes the undigested cell walls and tissues.
4. Centrifuge for 3 min at 100 Â g without brake. Remove
supernatant and resuspend the protoplasts by gentle swirling.
5. Add 5 mL of W5 buffer per tube. Gather the content of two
tubes into one. Centrifuge for 1 min at 100 Â g without brake.
6. Remove supernatant and softly resuspend protoplasts.
7. Repeat steps 5 and 6 until all protoplasts are gathered into two
tubes.
8. Gently resuspend protoplasts by inversion of the tubes and add
10 mL of MMG buffer per tube. Centrifuge for 1 min at
100 Â g without brake. Remove supernatant and softly resuspend protoplasts.
9. Add 2 mL of MMG, gather tubes, and incubate for at least
15 min on ice.
10. Add 10 mL of MMG. Centrifuge for 1 min at 100 Â g. Remove
supernatant and softly resuspend protoplasts in 1 or 2 mL
of MMG.
422
Ste ´ phanie Lalande et al.