Electric impulses (8 kV/cm, 5 microseconds) were found to increase greatly the uptake of DNA into cells. When linear or circular plasmid DNA containing the herpes simplex thymidine kinase (TK) gene is added to a suspension of mouse L cells deficient in the TK gene and the cells are then exposed to electric fields, stable transformants are formed that survive in the HAT selection medium. At 20 degrees C after the application of three successive electric impulses followed by 10 min to allow DNA entry there result 95 (+/‐ 3) transformants per 10(6) cells and per 1.2 micrograms DNA. Compared with biochemical techniques, the electric field method of gene transfer is very simple, easily applicable, and very efficient. Because the mechanism of DNA transport through cell membranes is not known, a simple physical model for the enhanced DNA penetration into cells in high electric fields is proposed. According to this ‘electroporation model’ the interaction of the external electric field with the lipid dipoles of a pore configuration induces and stabilizes the permeation sites and thus enhances cross membrane transport.

We report here the transformation of two species of orchid, Dendrobium phalaenopsis and D. nobile, by biolistic bombardment. Calli or protocorm-like bodies (PLBs) were used as target explants. Gold particles (1.0 µm) coated with plasmid DNA (pCAMBIA1301) encoding an intron-containing β-glucuronidase gene (gus-int) and a hygromycin phosphotransferase (hpt) gene were introduced into the PLBs or calli using the Bio-Rad PDS-1000/He Biolistic Particle Delivery System. Calli and PLBs were then chopped up and precultured in 1/2-strength MS medium supplemented with 0.4 M mannitol for a 1-h osmoticum treatment before bombardment. Immediately after bombardment, the calli and PLBs were transferred to 1/2-strength MS medium without mannitol for recovery. Putatively transformed plantlets were obtained by selection and regeneration on medium supplemented with 30 mg/l hygromycin. The highest efficiency of transformation was obtained when selection was conducted at 2 days post-bombardment. For D. phalaenopsis and D. nobile, respectively, about 12% and 2% of the bombarded calli or PLBs produced independent transgenic plants. Integration and expression of the transgenes were confirmed by Southern hybridization and Northern hybridization. No nontransformed plants were regenerated, indicating a tight selection scheme. However, separate incorporation of the gus gene and the hpt gene was observed, and in one transgenic line the gus gene was integrated into the genome of the transgenic plant, but not expressed.