( omonas reinhardtii with an a...)Sub-Micron Gold Particles Are Superior to Larger Particles for Efficient Biolistic Transformation of Organelles and Some Cells

omonas reinhardtii with an arginine-requiring point mutation in the ARG7 gene encoding argininosuccinate lyase (strain CC-48) or a non-photosynthetic, acetate-requiring deletion mutation in the atpB gene (strain CC-373) were used for assaying nuclear and chloroplast transformation, respectively. For nuclear transformation, CC-48 cells were bombarded with plasmid P-389 (pARG7.8; Debuchy, et al., 1989) containing the wild-type ARG7 argininosuccinate lyase gene from C. reinhardtii; for chloroplast transformation, CC-373 cells were bombarded with plasmid P-437 (BamH I chloroplast fragment 10 cloned into pBluescript KS+) containing the wild type atpB gene from C. reinhardtii. Bombardment conditions were as follows (Boynton & Gillham, 1993): 0.6 mg of 0.6 or 1 gold or M10 tungsten (average size = 0.7 ) particles were coated with 1.5 g of CsClpurified P-389 (for nuclear transformation) or P-437 plasmid (for chloroplast transformation); 4 x 10⁷ log-phase cells in 0.1% top agar were spread on 100 mm agar plates; 10 plates were shot for each experimental condition; cells were placed at Target Level 2; the bombardment chamber was reduced to 26 Hg vacuum and cells were bombarded at a helium pressure of 1,300 psi. Following bombardment, cells from each plate were respread onto two plates of selective media: nuclear transformants were re-plated onto arginine-free media and chloroplast transformants were re-plated onto acetate-free media.

For nuclear transformation, arg⁺ colonies appeared within 7 days post-bombardment, while photosynthetic colonies resulting from chloroplast transformation were visible 35 days postbombardment. In these experiments, final scoring of all transformants was performed
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