--J. Stadler and Huang H. C. Lin

The engulfing of foreign substances (phagocytosis or pinocytosis) by mammalian cells is a normal process involving plasma membrane (Berlin et al., Cell 15:327, 1978). We were curious to see if maize protoplasts would take up large particles since natural or induced phagocytosis of foreign DNA aggregates might eventually prove to be the least cytotoxic and most efficient method for direct gene transfer to these cells. To test the phagocytic ability of Black Mexican Sweet (line BMS-M) protoplasts, the cells were presented with Fluoresbrite-labeled, 100µ polystyrene latex beads (Polysciences) after removal of the cell wall (MNL 62:7, 1988). The latex beads were prepared by a 1:250 dilution with growth medium to yield approximately 109 beads in 1.0ml. One ml of the bead suspension was gently mixed with 106 protoplasts in 1.0ml growth medium with 8% mannitol (Somers et al., Plant Sci. 53:249, 1988) and incubated at 28 C without shaking. At various times after addition of beads a protoplast sample was removed and examined with a Leitz Dialux 20 microscope equipped with a Ploem illuminator and a filter combination designed for wide-band blue fluorescence. At least 100 protoplasts were examined to establish each data point.

The results of two preliminary experiments are shown in Table 1. In the first, labeled beads were added to the protoplasts immediately after cell wall removal, or 16h, or 24h later. Protoplasts were then examined microscopically 24h after the addition of the beads. When the beads were added at 0 time, 31% of the protoplasts took up an average of 8.0 beads, but when the beads were added to cells 16h after protoplasting only 9% took up the particles. No beads were taken up by the treated cells if they were presented 24h after cell wall removal. These data suggest that the latex particles are efficiently taken up until major portions of the cell wall are reformed by 16h.

Table 1. Uptake of 100 u polystyrene latex beads by maize (BMS-M) protoplasts.
 

	Cells with latex beads (%)	Average number of latex beads per cell
A. EXPERIMENT 1
Beads added at: 0h	31	8.0
Beads added at: 16h	9	3.4
Beads added at: 24h	0	0
(All cells examined 24h after
addition of beads.)
B. EXPERIMENT 2
(All beads added at 0h)
Cells examined at: 2h	23	2.0
6h	36	2.9
12h	31	4.5
24h	32	4.8

We do not yet have microscopic proof (by EM or counterstaining) that the brightly fluorescing 100µ beads are in the cells rather than accidentally attached to the surface of the protoplast. However, several observations have convinced us that most cell-associated beads have been phagocytized and do lie within the cells. First, very often the engulfed particles have been moved through the tonoplast and concentrated in the vacuole. Also, in time course experiments like the one just described (Table 1A) accidental association of the beads with the cell surface would be expected to give a high "background" at all times. However, this background is not seen when beads are added to 24h-old protoplasts and allowed to incubate for an additional 24h.

The second experiment (Table 1B) followed a time course for particle uptake when the beads were added immediately after cell wall removal. Twenty three percent of cells had taken in an average of 2 particles per cell by 2h. By 6h 30% of the cells had engulfed an average of 3 beads. The percentage of cells with beads remained the same between 6 and 24h although the average number of beads per cell increased slightly. These data again suggest that maize cell protoplasts are quite capable of phagocytosis of large 100µ polystyrene latex beads before cell-wall formation interferes. If so, the cells should probably also be capable of the endocytosis of large DNA aggregates.

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