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Optimization of Biolistic Transformation Using the Helium-Driven PDS-1000/He System

microparticle and particle accelerator parameters with Bio-Rad's PDS-1000/He system using two biological systems, transient expression of the -glucuronidase (GUS) gene in cauliflower epidermal cortex, and stable expression of a wild-type URA 3 gene in an auxotrophic mutant of Saccharomyces cerevisiae. The microparticle variables include the size, amount, and type of microparticles; the accelerator parameters include the helium pressure, the distance between the rupture disk and macrocarrier (the gap distance), the distance between the macroprojectile and the stopping screen (the macrocarrier travel distance), and the distance between the stopping screen and the biological target (the target distance). See Figure 1 for a graphic representation of these variable distances. In order to obtain the maximum number of transformants, it is also necessary to optimize the biological parameters associated with the target tissue. These factors include the state of growth of the target cells, the cell density, the osmolarity of the bombardment medium, the treatment of the cells post-bombardment, and the expression vector being used for transformation (Sanford, et al., 1992).


Methods
Preparation of Yeast and Cauliflower
The yeast auxotroph, S. cerevisiae st 948 (Armaleo, et al., 1990), contains non-reverting mutations in the ura 3 and leu 2 genes, and was used for all experiments with yeast. Yeast were prepared for bombardment as described by Klein, et al. (1991). Briefly, cells were grown to early stationary growth phase in YEPD media. Cells were concentrated 100-fold by centrifugation, and 108 cells were spread on uracil-deficient agar plates containing 0.75
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