( NA and 1.6 g of YEp352 plasm...)Sub-Micron Gold Particles Are Superior to Larger Particles for Efficient Biolistic Transformation of Organelles and Some Cells

NA and 1.6 g of YEp352 plasmid; 2 x 10⁸ cells were spread on 100 mm agar plates; 5 plates were shot for each experimental condition; the bombardment chamber was reduced to 28 Hg vacuum and cells were bombarded at Target Level 2 at a helium pressure of 1,1001,300 psi.

Nuclear transformation was quantitated by counting Ura⁺ transformants 5 days post-bombardment following growth of transformed cells on uracil-deficient media. Ura⁺ transformants were then analyzed for mitochondrial transformation by mating to S. cerevisiae strain 161 on glucose-containing media (Anziano & Butow, 1991; Butow, et al., 1996). Mitochondrial transformants were identified by replica plating these colonies onto media containing glycerol. Results are summarized in Table 4. Nuclear transformation is quite efficient using either 0.6 gold particles or M5 tungsten to deliver the DNA; there is a greater than 10- fold decrease in the number of transformants when 1 gold particles are substituted for 0.6 gold particles. In these experiments, mitochondrial transformants were only isolated when sub-micron sized gold particles were used. While mitochondrial transformants have been isolated using larger tungsten particles, results with those particles were extremely erratic in our hands, even in terms of nuclear transformation. Similar results were found when bombardment was performed with the Ura⁺ and COXI sequences on a single plasmid (results not shown).

Conclusions
Results presented here show that particle size is an important factor for optimizing Biolistic transformation. Maximum stable transformation of cellular organelles (chloroplasts and mitochondria
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