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Sub-Micron Gold Particles Are Superior to Larger Particles for Efficient Biolistic Transformation of Organelles and Some Cells

1314 days after bombardment. Results of several bombardments are summarized in Table 3. Both nuclear and chloroplast transformation efficiencies were on average 2.5-fold higher using 0.6 gold particles compared to using M10 tungsten particles. In a single experiment, nuclear transformation of Chlamydomonas was about two-fold lower using 1 gold particles than using 0.6 gold particles, while the level of Chlamydomonas chloroplast transformation was four-fold lower using 1 gold particles than using 0.6 gold particles.

Stable transformation of yeast mitochondria. A derivative of Saccharomyces cerevisiae strain MCC109 (MATα ade2-101 ura3-52 kar1-1) lacking mitochondrial DNA [rhoo] was used for assaying nuclear and mitochondrial transformation. Cells were bombarded with particles carrying the URA3 gene on plasmid YEp352 and a plasmid carrying intron 2 from the COXI gene (either pJVM161 or pJVM164; Moran, et al., 1995) and Ura+ transformants were selected on media lacking uracil. Mitochondrial transformants were identified by mating the [rhoo] Ura+ nuclear transformants with a respiration-deficient mutant (S. cerevisiae strain 161) containing a deletion within intron 2 of the COXI gene (Zimmerly, et al., 1995) and screening for progeny capable of respiring on glycerol-containing media. Recombination between the defective portion of the COXI gene present in S. cerevisiae strain 161 and the COXI gene from the plasmid present in the mitochondria of the transformant restores respiratory growth. Bombardment conditions were as follows: 0.6 mg of gold or M5 tungsten (average size = 0.4 ) were coated with a mixture of 8 g of COXI mitochondrial plasmid D
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