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Cotyledon explants were established from 5- to 6-day-old `Minilee' seedlings germinated on MS medium with 2% sucrose and 0.7% TC agar, and precultured for 2 days on modified MS as above but with 3% sucrose and 5 μM BA before bombardment. Plasmid DNA [pUC221 and pUC472, which contain the β-glucuronidase (GUS) and NPTII (kanamycin resistance) genes, respectively] was delivered to the explants using a modified particle inflow gun following precipitation on to 1.1 μm tungsten particles. Explants infected with Agrobacterium (strain LBA4404 with the binary vector pBI121, which contains genes for GUS and kanamycin resistance) were not precultured prior to cocultivation for 4 days. GUS expression was measured 1 week after transfer to selection medium. Infection with Agrobacterium was the best method for delivering foreign DNA to watermelon cotyledons. Up to 300 GUS-expressing colonies were observed per explant following infection with Agrobacterium compared to 128 after bombardment.
Design modification of a particle inflow-type gun for particle bombardment significantly simplifies construction and reduces fabrication time. The gun consists of a high-speed electric solenoid valve mounted on and through a vacuum jar. DNA-coated tungsten particles are placed on the support grid of a filter housing and accellerated by a burst of pressurized helium, which is controlled by a timer. Specimens are held between plastic screens and their distance from the particle support grid is adjusted with a miniature laboratory apparatus positioner. Transient expression of GUS in cantaloupe cotyledons and grape somatic embryos was equivalent to that obtained with a conventional particle inflow gun. The device was constructed with locally-available hardware in 40 minutes using a hand drill, some thread taps and a thread die.