Figure 2.

Characterization of embryogenic and nonembryogenic protoplast-derived alfalfa cells formed under different conditions. A, Cell size expressed as the average of the length and width of the cells. Thirty cells were measured per treatment. B, Increase of ascorbate peroxidase activity indicating oxidative stress response of the cells due to excess Fe or 2,4-D in the medium. C, Cell division rate determined under a light microscope as the percentage of cells already divided at least once (at least 500 cells were counted). D, S phase progression followed by 5-bromo-2′-deoxyuridine (BrdU) incorporation into the nuclei of the cells during 36, 42, and 52 h of culture, respectively. E, The pH of the medium (pHe) at 0, 2, 3, and 4 d of culture. F, pHc of protoplast-derived cells grown under different conditions. Average pHc of 15 randomly selected cells. The pH values were determined using fluorescence microscopy following FDA staining and an in vitro calibration curve (see “Materials and Methods”). G, Vacuolar pH of protoplast-derived cells at 4 d of culture grown under different conditions. Average vacuolar pH of 15 randomly selected cells. The pH values were determined using fluorescence microscopy following BCECF-AM staining and an in vitro calibration curve (see “Materials and Methods”). Data are from at least three independent experiments. The se of the measurements is indicated on the bars. d.i.c., Days in culture; h.i.c., hours in culture; nd, no data.