( esults Stable transformat...)Sub-Micron Gold Particles Are Superior to Larger Particles for Efficient Biolistic Transformation of Organelles and Some Cells

esults
Stable transformation of Type I callus of Zea mays. Maize Type I callus (Wan, et al., 1995) was subjected to osmotic pretreatment by placing the tissue on high sucrose media for 4 hr. prior to bombardment. Tissues were bombarded in the PDS- 1000/He with 1 or 0.6 gold particles coated with pBC17 (Goff, et al., 1990), a plasmid which encodes genes for the C1 regulatory gene and the B-Peru allele of the B regulatory gene in the maize anthocyanin biosynthetic pathway. When expressed together, these genes activate this pathway and give rise to pigmented cells. Bombardment conditions were as follows. Each plate was shot twice. Each shot contained 0.06 mg or 0.5 mg of 0.6 gold particles, or 0.5 mg of 1 gold particles, and all particles were coated with 0.33 g of plasmid pBC17; 16 target tissue pieces were placed in a ring on a 100 mm plate; 3 plates were shot per treatment; a stainless steel 200 x 200 wire mesh screen (McMaster-Carr, cat. no. 9236T11) was inserted at Level 1 as a post-launch baffle (Russell, et al., 1992) to reduce cell damage and the tissue sections were placed at Target Level 2; the bombardment chamber was reduced to 28 Hg vacuum and cells were bombarded at a helium pressure of 650 or 900 psi.

Transient expression and target tissue damage were qualitatively assessed 4 days after gene delivery. Generally, bombardment with 1 mg of 1 gold particles produced relatively high levels of transient expression as judged by observation of pigmented cells. The level of transient expression in cells bombarded with 0.6 gold particles was dependent on the amount of gold used in bombardment. Reducing the amount of gold eight-fold, from 1 mg to 0.12 mg, resulted in an eightfold decre
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