The uncut plasmid DNA pAHC25 [32 (link)] was used only for transient expression analysis in suspension cells which were treated 4 days after subculture. Five ml of well-mixed suspension adjusted to OD = 0.1–0.2 was spread on Ruled Qualitative Filter Paper discs with a 5 mm Ruled Grid, diameter 55 mm (Whatman, USA). A vacuum was applied to get an even spread of a very thin layer of cells on the surface of the filter paper. The coated papers were subsequently transferred to MS2–2,4D medium with 100 g/L sucrose for osmotic pre-treatment for 4 h. Bombardment conditions for the callus tissues and the suspension cells were the same as described above. The transformed cells were maintained on the MS2 medium for 48 h. GUS staining with X-gluc (0.5 mg/mL) was as described in [33 (link)]. Filter disks with the treated cells were carefully transferred into 60 mm plastic Petri dishes with 550 μl of the X-gluc solution and incubated overnight at 37 °C. For further details see [34 (link)].
To quantify GUS expression in the T. monococcum suspension cells, the number of cells with blue staining was counted on the same Ruled Qualitative Filter Papers under a Binocular microscope (Nikon, Japan), and recorded as described earlier [34 (link), 35 ]. Each treatment was repeated at least three times, with mean and variability calculated.
To quantify GUS expression in the T. monococcum suspension cells, the number of cells with blue staining was counted on the same Ruled Qualitative Filter Papers under a Binocular microscope (Nikon, Japan), and recorded as described earlier [34 (link), 35 ]. Each treatment was repeated at least three times, with mean and variability calculated.