( lasmid pBI221 and stained fo...)Optimization of Biolistic Transformation Using the Helium-Driven PDS-1000/He System

lasmid pBI221 and stained for GUS-expressing cells is shown in Figure 2. Each section in the array was scored separately and given a semi-quantitative score based on the relative number of GUS-positive cells (see Figure 2 and Table 1, footnote c). Results of an experiment to determine the effects of microparticle size, helium pressure, and target distance are shown in Table 1. Several observations were made from the initial experiment (Table 1, Experiment 1): (1) while both M10 (median particle diameter of 0.7 mm) and M17 tungsten microparticles may be used for cauliflower transformation, the latter are consistently more effective; (2) tissue sections bombarded at level 2 (6 cm target distance) are transformed more efficiently than sections bombarded at level 4 (12 cm target distance); and, (3) changing the helium pressure between 1,100 and 1,550 psi has little effect on transformation efficiency. Subsequent results confirmed these conclusions and also indicated that similar transformation efficiencies were achieved with all pressures from 900 to 1,550 psi (Table 1, Experiments 2 and 3). Transformation results using 1.0 mm and 1.6 mm gold microparticles to bombard tissue sections at level 2 or 4 also indicated that the highest transformation efficiency was obtained using the larger particles with the shorter target distance. As with the tungsten microparticles, varying the helium pressure between 900 and 1,550 psi had little effect on transformation efficiency.

The effect of changing the gap width and macrocarrier travel distance on GUS expression was assayed using optimal conditions determined in the initial experiments (Table 2). At a target distance of 6 cm and bombardment press
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