Effects of DNA and PEG concentrations on transfection efficiency.

<p>Oil palm protoplasts were transfected with 25 µg (A) or 50 µg (B) of CFDV-hrGFP plasmid DNA, and with 50 µg CFDV-hrGFP plasmid DNA in the presence of 25% (C), 40% (D) or 50% PEG (E). Black arrows indicate damaged protoplasts caused by PEG toxicity. The transfection efficiencies represent the mean of three replicates. Scale bar  = 10 µm in (A) and (B), 25 µm in (C)–(F).</p

Effect of DNA concentration on transformation efficiency.

<p>Protoplasts were injected with 100/µl (A and B), 500 ng/µl (C and D) and 1000 ng/µl (E and F) DNA solutions and monitored for evidence of GFP fluorescence after culture for one month. The transformation efficiencies represent the mean of three replicates. Arrows indicate the surviving injected protoplasts and small dots indicate dead cells. Scale bar  = 100 µm. All cells were injected in the visial field shown in this figure, but uninjected cells also developed into (non-fluorescent) microcalli.</p

Microinjection of DNA into oil palm protoplasts.

<p>Oil palm protoplasts were isolated from a 3-month-old cell suspension culture after subculture for 7 days, mixed with 1% alginate solution in Y3A medium and distributed as a thin layer onto supporting medium (A). The embedded protoplasts were arranged in a single planar layer as confirmed by using the 10× objective (B). The protoplasts were incubated at 28°C in the dark for 3 days (C), and then placed on the microscope stage for DNA microinjection (D). The DNA solution was injected into the protoplast (E) and confirmed by Lucifer yellow fluorescence (F). GFP fluorescence was detected in the cytoplasm after 3 days (G and H). The injected protoplast is indicated by an arrow. Scale bar  = 1 cm in (A), (C) and (D), 100 µm in (B), 25 µm in (E)–(H).</p

Transfection efficiency is affected by different concentrations of MgCl₂ and the DNA incubation time.

<p>Oil palm protoplasts were transfected with 10 µg CFDV-hrGFP plasmid using 40% (w/v) PEG solution with MgCl₂ at concentrations of 10 mM (A), 25 mM (B), 50 mM (C) and 100 mM (D). Oil palm protoplasts were incubated with 10 µg CFDV-hrGFP plasmid DNA for 15 min (E) or 30 min (F), and then mixed with PEG-MgCl₂ solution. Transfection efficiency was calculated as the number of GFP-fluorescent protoplasts divided by the total number of protoplasts in one representative microscope field. The transfection efficiencies represent the mean of three replicates. Scale bar  = 10 µm in (A)–(E), 75 µm in (F).</p

Oil palm protoplasts showing GFP fluorescence.

<p>A 3-month-old oil palm cell suspension culture in Y35N5D2iP liquid medium (A) was collected and cultured on Y35N5D2iP solid medium (B) for protoplast isolation (C). Transient GFP fluorescence was observed in protoplasts isolated from the 3-month-old cell suspension culture after subculture for 7 days (D) and 14 days (E), and protoplasts isolated from the 4-month-old cell suspension culture (F). CLSM images are shown representing GFP fluorescence (GFP), autofluorescence (Auto) and bright field (Bright) as well as three-layer images (Merged) of the protoplasts. Red arrows indicate autofluorescence. Scale bar  = 1 cm in (A) and (B), 100 µm in (C), 10 µm in (D), 25 µm in (E) and (F).</p

Development of microcalli expressing GFP.

<p>Five days after DNA microinjection, the alginate layer was transferred to Y3A liquid medium comprising 5.5% (w/v) sucrose and 8.2% (w/v) glucose supplemented with 10 µM NAA, 2 µM 2,4–D, 2 µM IBA, 2 µM GA₃, 2 µM 2iP and 200 mg/l ascorbic acid and cultured at 28°C for 2 weeks. The medium was then replaced with similar Y3A liquid medium comprising 4% (w/v) sucrose and 7.2% (w/v) glucose to allow the development of microcolonies (A and B, after 2 months). The medium was then replaced with Y3A liquid medium comprising 4% (w/v) sucrose to promote the conversion of microcolonies (C and D, after 4 months) into microcalli (E and F, after 6 months). Finally, the alginate layer containing microcalli (G) was transferred onto Y31N0.1BA solid medium (H) for the regeneration of oil palm plants. Arrows indicate the injected protoplasts. The transformation efficiencies represent the mean of three replicates. Scale bar  = 100 µm in (A)–(F), 1 cm in (G) and (H).</p