( M sorbitol and 0.75 M mannitol. S...)Optimization of Biolistic Transformation Using the Helium-Driven PDS-1000/He System

M sorbitol and 0.75 M mannitol. Seeded plates were kept for 2-4 hours at room temperature before transformation. After bombardment, the cells were incubated for 60 hours at 30 C, at which time the colonies were counted.

Cauliflower was obtained from a local produce market. The main stems were sectioned into approximately 1 cm squares (2-4 mm thick), and nine longitudinal sections were placed in a 3 x 3 array in the center of 100 mm Petri plates containing 1% agar (Klein, et al., 1991). The agar acted as a support to help the tissue sections absorb the shock from the bombardment and also kept the tissues moist during overnight incubation. After bombardment of the epidermal cells, the tissue was incubated for 24 hours at 24 C. The location of GUS-expressing cells was visualized using a histochemical reaction (Jefferson, et al., 1987). Cauliflower sections were transferred to 16 mm wells containing 0.8 ml of GUS assay buffer (0.5 mg/ml 5-bromo-4-chloro-3-indoyl--D-glucuronic acid, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 0.06% Triton X-100, 0.1 M sodium phosphate buffer, pH 7.0) and incubated at 37 C. After 18 hours, the tissue sections were evaluated for their level of GUS expression (see Table 1, footnote c).

Plasmids
Plasmid YEp352 (Hill, et al., 1986), which contains a wild-type ura 3 gene, was used for transformation of yeast. Cauliflower tissue was bombarded with the plasmid pBI221, which contains the GUS gene expressed from the cauliflower mosaic virus 35S promoter (Jefferson, et al., 1987). Both plasmids were prepared by alkaline lysis (Maniatis, et al., 1982) followed by cesium chloride purification and ethanol precipitation.

Biolistic Transformat
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