For electroporation experiments, protoplasts were isolated from five-day old Nicotiana tabacum BY-2 suspension cells. Suspension cultures were grown at 23°C with shaking (130 rpm) in a medium containing Murashige and Skoog salts [21 (link)] supplemented with 1 mg/L thiamine-HCl, 370 mg/L KH2PO4, 30 g/L sucrose, and 2 mg/L2, 4-dichlorophenoxyacetic acid, pH 5.7. Cells were sub-cultured once per week by adding 2.5–3 ml of inoculum to 50 ml of fresh medium in 250 ml Erlenmeyer flasks. The 50 ml of suspension culture was centrifuged at 250 × g at room temperature for 5 min in a Sorvall GLC-2 centrifuge, and the pellet (15 ml packed cell volume) was re-suspended in 50 ml of protoplast isolation solution containing 7.4 g/L CaCl2 ·2H2O, 1 g/L NaOAc, and 45 g/L mannitol supplemented with 1.2% cellulose R10 (Onazuka) and 0.6% Macerozyme (Duchefa), pH 5.7. Approximately 15 ml of suspension culture was transferred into three 20 × 100 mm sterile Petri dishes and incubated in the dark with a gentle shaking (40 rpm) at room temperature for 4 hours. The protoplasts were washed twice with protoplast isolation solution and the pellet was re-suspended in 50 ml of floating solution (99 mg/L myo-inositol, 2.88 g/L L-proline, 100 mg/L enzymatic casein hydrolysate, 102.6 g/L sucrose, 97.6 mg/L MES buffer, 4.3 g/L MS salts, 1 mg/L thiamine-HCl, 370 mg/L KH2PO4, pH 5.7). Protoplasts (floating on the top of the solution) were transferred to a new tube, washed twice, and suspended in 50 ml of electroporation solution (10 mM NaCl, 4 mM CaCl2·2H2O, 120 mM KCl, 10 mM HEPES, 0.6 M mannitol, pH 7.2). Cells were incubated at 42°C for 5 min and kept in ice for 10 min before electroporation. Aliquots of protoplasts containing approximately 3 × 106 cells/ml were used for electroporation. ~20 μg of each plasmid DNA were added to 300 μl of protoplasts in a tube and placed on ice. The electroporation was conducted using a BioRad Gene Pulser apparatus at 0.16 kV, with the Pulse Controller set to infinity and the capacitance extender set to 960 μFD. After 10 min incubation on ice, the protoplasts were transferred into 10 ml of BY-2 culture medium supplemented with 0.4 M mannitol and incubated overnight.
For direct DNA uptake experiments, 20 ml BY-2 suspension cells were transferred to a sterile conical centrifuge tube and centrifuged at 3000 rpm for 10 min. The pelleted cells were suspended in 10–20 ml protoplast digestion enzyme solution (1.2% Cellulase Onozuka RS [Duchefa] and 0.6% Macerozyme R-10 [Duchefa] in 10 mM CaCl2·2H2O, 12 mM NaOAc, 11% mannitol, pH 5.7) and incubated in the dark with shaking (40 rpm) for 3–4 hr at room temperature. The protoplasts were filtered through 40 μm nylon mesh and centrifuged in a 50 ml conical tube at 250 × g for 5 min. The protoplasts were collected and suspended in 10 ml protoplast floating solution (per liter: 99 mg myo-inositol, 2.88 g L-proline, 100 mg enzymatic casein hydrolysate, 102.6 g sucrose, 97.6 mg MES buffer, 4.3 g MS salts, 1 mg Vitamin B1, 370 mg KH2PO4, pH 5.7) and centrifuged at 250 × g for 10 min. Protoplasts floating in this solution were removed and 10 ml W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES, pH 5.7) was added. The solution was centrifuged at 250 × g for 5 min, and the protoplasts pelleted. Protoplast concentration was adjusted to 1 × 106/ml and the solution incubated on ice for 30 min. The protoplasts were again centrifuged at 250 × g for 5 min and suspended at a density of 1 × 106 cells/ml in MMg solution (0.6 M mannitol, 15 mM MgCl2, 4 mM MES, pH 5.7). DNA (10 μg in 10 μl) was added to 100 μl protoplasts, followed by addition of 110 μl PEG solution (per ml: 0.4 g PEG 4000 [Fluca], 0.6 ml 1 M mannitol, 100 μl 1 M CaCl2), and the protoplasts incubated at room temperature for 30 min. After addition of 1 ml W5 solution, the protoplast suspension was centrifuged at 250 × g for 5 min. The protoplast pellet was suspended in 1 ml incubation solution (per liter: MS salts and vitamins, 2 mg/L 2,4-D, 3% sucrose, 0.4 ~ 0.6 M mannitol) and incubated in the dark for 16 hr at 26°C.
Particle bombardment of onion epidermal peel layers was carried out using a Biolistic Particle Delivery System (Bio-Rad) PDS-1000. Samples (whole onion from which the dry outer layer was removed) were sterilized in ~300 ml 2% NaOCl and 2–3 drops Tween-20 for 15 min. The tissue was washed with sterilize H2O at least five times. The upper epidermal layer of the onion was removed, cut into 2 × 2 cm squares, and placed on a plate containing 1/2 MS medium. 5 μg of each plasmid DNA was used in all experiments. Gold particles size was 1.6 μm (INBIO GOLD). 0.15–0.2 mg particles/per shot were used with a chamber vacuum of 27 in Hg. Particles were accelerated with a pressure of 1100 psi. The distance between the projectile source and the samples was 6 cm.
For direct DNA uptake experiments, 20 ml BY-2 suspension cells were transferred to a sterile conical centrifuge tube and centrifuged at 3000 rpm for 10 min. The pelleted cells were suspended in 10–20 ml protoplast digestion enzyme solution (1.2% Cellulase Onozuka RS [Duchefa] and 0.6% Macerozyme R-10 [Duchefa] in 10 mM CaCl2·2H2O, 12 mM NaOAc, 11% mannitol, pH 5.7) and incubated in the dark with shaking (40 rpm) for 3–4 hr at room temperature. The protoplasts were filtered through 40 μm nylon mesh and centrifuged in a 50 ml conical tube at 250 × g for 5 min. The protoplasts were collected and suspended in 10 ml protoplast floating solution (per liter: 99 mg myo-inositol, 2.88 g L-proline, 100 mg enzymatic casein hydrolysate, 102.6 g sucrose, 97.6 mg MES buffer, 4.3 g MS salts, 1 mg Vitamin B1, 370 mg KH2PO4, pH 5.7) and centrifuged at 250 × g for 10 min. Protoplasts floating in this solution were removed and 10 ml W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES, pH 5.7) was added. The solution was centrifuged at 250 × g for 5 min, and the protoplasts pelleted. Protoplast concentration was adjusted to 1 × 106/ml and the solution incubated on ice for 30 min. The protoplasts were again centrifuged at 250 × g for 5 min and suspended at a density of 1 × 106 cells/ml in MMg solution (0.6 M mannitol, 15 mM MgCl2, 4 mM MES, pH 5.7). DNA (10 μg in 10 μl) was added to 100 μl protoplasts, followed by addition of 110 μl PEG solution (per ml: 0.4 g PEG 4000 [Fluca], 0.6 ml 1 M mannitol, 100 μl 1 M CaCl2), and the protoplasts incubated at room temperature for 30 min. After addition of 1 ml W5 solution, the protoplast suspension was centrifuged at 250 × g for 5 min. The protoplast pellet was suspended in 1 ml incubation solution (per liter: MS salts and vitamins, 2 mg/L 2,4-D, 3% sucrose, 0.4 ~ 0.6 M mannitol) and incubated in the dark for 16 hr at 26°C.
Particle bombardment of onion epidermal peel layers was carried out using a Biolistic Particle Delivery System (Bio-Rad) PDS-1000. Samples (whole onion from which the dry outer layer was removed) were sterilized in ~300 ml 2% NaOCl and 2–3 drops Tween-20 for 15 min. The tissue was washed with sterilize H2O at least five times. The upper epidermal layer of the onion was removed, cut into 2 × 2 cm squares, and placed on a plate containing 1/2 MS medium. 5 μg of each plasmid DNA was used in all experiments. Gold particles size was 1.6 μm (INBIO GOLD). 0.15–0.2 mg particles/per shot were used with a chamber vacuum of 27 in Hg. Particles were accelerated with a pressure of 1100 psi. The distance between the projectile source and the samples was 6 cm.
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