3.5 Isolation
of Protoplasts from
Cell Suspension
Cultures
Protoplasts can be conveniently isolated from Arabidopsis cell suspension cultures, every week and with high yields, high percentage
of vitality, and very good reproducibility [6]. They can be obtained
from wild-type cultures and then subjected to transient transformation [12], or from transgenic lines stably expressing proteins of
interest (Fig. 4).
1. Transfer 2.5 mL PCV of a mid-exponential phase (4- to 5-day-old) (see Subheading 3.2.5) cell suspension culture into a sterile
50 mL tube (see Note 26).
2. Add 10 mL Enzymatic Solution (see Subheading 2.8) to the
tube by using the pipet-aid and a sterile disposable 10 mL
pipette.
3. Wrap the tube in aluminum foil and incubate for 2 h horizontally on the orbital shaker set at 80 rpm in the growth chamber.
4. At the end of the incubation time, in the laminar flow hood
transfer the cell suspension on top of a sterile 50 μm concave
nylon mesh, mounted on a 100 mL glass beaker thanks to a
rubber band (see Note 27).
5. Let the solution completely leach through the nylon mesh,
gently helping the process with the tip of a sterile disposable
plastic Pasteur pipette.
6. Cut the rubber band with a sterile scalpel and discard the nylon
mesh with the undigested cell debris on top.
7. Use a new sterile disposable plastic Pasteur pipette to transfer
the filtered protoplast solution in a sterile 15 mL tube.
8. Centrifuge the tube at 60 g for 5 min at room temperature,
brake off.
9. Discard the supernatant by using a sterile disposable plastic
Pasteur pipette.
10. Add 5 mL of Protoplast Resuspension Buffer (see Subheading
2.8) to the tube and gently resuspend the pellet by slowly
swinging the tube.
11. Repeat centrifugation and washes twice.
12. Centrifuge the tube again, then resuspend the pellet in 1 mL
fresh Protoplast Resuspension Buffer (see Note 28) (Fig. 4).
4 Notes
1. Large volumes of liquid medium, such as 3 or 5 L, can be
prepared. Remember to clearly label bottles containing different culture media, possibly with different colored tapes, to
avoid confusion when you use them. Before use, gently shake
the bottle containing the liquid medium in order to mix the
180
Enrico Cortese et al.
of Protoplasts from
Cell Suspension
Cultures
Protoplasts can be conveniently isolated from Arabidopsis cell suspension cultures, every week and with high yields, high percentage
of vitality, and very good reproducibility [6]. They can be obtained
from wild-type cultures and then subjected to transient transformation [12], or from transgenic lines stably expressing proteins of
interest (Fig. 4).
1. Transfer 2.5 mL PCV of a mid-exponential phase (4- to 5-day-old) (see Subheading 3.2.5) cell suspension culture into a sterile
50 mL tube (see Note 26).
2. Add 10 mL Enzymatic Solution (see Subheading 2.8) to the
tube by using the pipet-aid and a sterile disposable 10 mL
pipette.
3. Wrap the tube in aluminum foil and incubate for 2 h horizontally on the orbital shaker set at 80 rpm in the growth chamber.
4. At the end of the incubation time, in the laminar flow hood
transfer the cell suspension on top of a sterile 50 μm concave
nylon mesh, mounted on a 100 mL glass beaker thanks to a
rubber band (see Note 27).
5. Let the solution completely leach through the nylon mesh,
gently helping the process with the tip of a sterile disposable
plastic Pasteur pipette.
6. Cut the rubber band with a sterile scalpel and discard the nylon
mesh with the undigested cell debris on top.
7. Use a new sterile disposable plastic Pasteur pipette to transfer
the filtered protoplast solution in a sterile 15 mL tube.
8. Centrifuge the tube at 60 g for 5 min at room temperature,
brake off.
9. Discard the supernatant by using a sterile disposable plastic
Pasteur pipette.
10. Add 5 mL of Protoplast Resuspension Buffer (see Subheading
2.8) to the tube and gently resuspend the pellet by slowly
swinging the tube.
11. Repeat centrifugation and washes twice.
12. Centrifuge the tube again, then resuspend the pellet in 1 mL
fresh Protoplast Resuspension Buffer (see Note 28) (Fig. 4).
4 Notes
1. Large volumes of liquid medium, such as 3 or 5 L, can be
prepared. Remember to clearly label bottles containing different culture media, possibly with different colored tapes, to
avoid confusion when you use them. Before use, gently shake
the bottle containing the liquid medium in order to mix the
180
Enrico Cortese et al.