NAA and 50 μL of 1 mg/mL kinetin. Transfer to a cylinder and
adjust volume to 1 L with water. Autoclave and store at room
temperature.
2. Seed sterilization solution: 0.1% (v/v) Tween-20, 70% (v/v)
ethanol. Under a sterile hood, filter solution with a 0.20 μm
filter in a sterile bottle. Store at room temperature.
3. Seedling culture medium: 2.21 g/L MS222, 0.5 g/L 2-(Nmorpholino)ethanesulfonic acid (MES), 10 g/L sucrose,
0.68% agar, pH 5.7. Weigh 2.21 g of MS222, 0.5 g of MES
monohydrate, and 10 g of sucrose. Transfer to a glass beaker.
Add about 500 mL of water. Mix well and adjust pH to 5.7
with 1 N KOH. Transfer to a cylinder and adjust volume to 1 L
with water. Transfer to a bottle and add 6.8 g of agar. Autoclave
and store at room temperature.
4. 2 M KCl: Store at room temperature.
5. 1 M MgCl 2 : Store at room temperature.
6. 1 M CaCl 2 : Store at room temperature.
7. 5 M NaCl: Store at room temperature.
8. 0.5 M Mannitol, pH 5.7: Weigh 227.75 g of D-mannitol and
1.75 g of MES monohydrate. Transfer to a glass beaker. Add
about 1.5 L of water. Mix and adjust pH to 5.7 with 10 N
KOH. Transfer to a cylinder and adjust volume to 2.5 L with
water. Autoclave and store at 4
C.
9. 0.5 M MES, pH 5.7: Weigh 53.3 g of MES monohydrate and
transfer to a glass beaker. Add about 400 mL of water. Mix and
adjust pH to 5.7 with 10 N KOH. Transfer to a glass cylinder
and adjust volume to 500 mL with water. Autoclave and store
at room temperature.
10. MM buffer: 0.5 M Mannitol, 4 mM MES, pH 5.7. Pipette
8 mL of 0.5 M MES, pH 5.7, and transfer to a 1 L glass
cylinder. Add 0.5 M mannitol, pH 5.7, to a final volume of
1 L and mix well. Autoclave and store at 4
C.
11. MMG buffer: 0.5 M Mannitol, 4 mM MES, 15 mM MgCl 2 ,
pH 5.7. Pipette 7.5 mL of 1 M MgCl 2 and transfer to a 1 L
glass cylinder. Add MM buffer to a final volume of 500 mL and
mix well. Autoclave and store at 4
C.
12. WI buffer: 0.5 M Mannitol, 4 mM MES, 20 mM KCl, pH 5.7.
Pipette 5 mL of 2 M KCl and transfer to a 1 L glass cylinder.
Add MM buffer to a final volume of 500 mL and mix well.
Autoclave and store at 4
C.
13. W5 buffer: 2 mM MES, 154 mM NaCl, 125 mM CaCl 2 , 5 mM
KCl, pH 5.7. Mix in a glass cylinder 62.5 mL of 1 M CaCl 2 ,
15.4 mL of 5 M NaCl, 2 mL of 0.5 M MES pH 5.7, and
1.25 mL of 2 M KCl. Add water to a final volume of 500 mL
and mix well. Autoclave and store at 4
C.
Plant Protoplasts-Based System to Transfect RNA
415
adjust volume to 1 L with water. Autoclave and store at room
temperature.
2. Seed sterilization solution: 0.1% (v/v) Tween-20, 70% (v/v)
ethanol. Under a sterile hood, filter solution with a 0.20 μm
filter in a sterile bottle. Store at room temperature.
3. Seedling culture medium: 2.21 g/L MS222, 0.5 g/L 2-(Nmorpholino)ethanesulfonic acid (MES), 10 g/L sucrose,
0.68% agar, pH 5.7. Weigh 2.21 g of MS222, 0.5 g of MES
monohydrate, and 10 g of sucrose. Transfer to a glass beaker.
Add about 500 mL of water. Mix well and adjust pH to 5.7
with 1 N KOH. Transfer to a cylinder and adjust volume to 1 L
with water. Transfer to a bottle and add 6.8 g of agar. Autoclave
and store at room temperature.
4. 2 M KCl: Store at room temperature.
5. 1 M MgCl 2 : Store at room temperature.
6. 1 M CaCl 2 : Store at room temperature.
7. 5 M NaCl: Store at room temperature.
8. 0.5 M Mannitol, pH 5.7: Weigh 227.75 g of D-mannitol and
1.75 g of MES monohydrate. Transfer to a glass beaker. Add
about 1.5 L of water. Mix and adjust pH to 5.7 with 10 N
KOH. Transfer to a cylinder and adjust volume to 2.5 L with
water. Autoclave and store at 4
C.
9. 0.5 M MES, pH 5.7: Weigh 53.3 g of MES monohydrate and
transfer to a glass beaker. Add about 400 mL of water. Mix and
adjust pH to 5.7 with 10 N KOH. Transfer to a glass cylinder
and adjust volume to 500 mL with water. Autoclave and store
at room temperature.
10. MM buffer: 0.5 M Mannitol, 4 mM MES, pH 5.7. Pipette
8 mL of 0.5 M MES, pH 5.7, and transfer to a 1 L glass
cylinder. Add 0.5 M mannitol, pH 5.7, to a final volume of
1 L and mix well. Autoclave and store at 4
C.
11. MMG buffer: 0.5 M Mannitol, 4 mM MES, 15 mM MgCl 2 ,
pH 5.7. Pipette 7.5 mL of 1 M MgCl 2 and transfer to a 1 L
glass cylinder. Add MM buffer to a final volume of 500 mL and
mix well. Autoclave and store at 4
C.
12. WI buffer: 0.5 M Mannitol, 4 mM MES, 20 mM KCl, pH 5.7.
Pipette 5 mL of 2 M KCl and transfer to a 1 L glass cylinder.
Add MM buffer to a final volume of 500 mL and mix well.
Autoclave and store at 4
C.
13. W5 buffer: 2 mM MES, 154 mM NaCl, 125 mM CaCl 2 , 5 mM
KCl, pH 5.7. Mix in a glass cylinder 62.5 mL of 1 M CaCl 2 ,
15.4 mL of 5 M NaCl, 2 mL of 0.5 M MES pH 5.7, and
1.25 mL of 2 M KCl. Add water to a final volume of 500 mL
and mix well. Autoclave and store at 4
C.
Plant Protoplasts-Based System to Transfect RNA
415