© 1996 Oxford University Press
RESEARCH-ARTICLE |
Expression of foreign genes, GUS and hygromycin resistance, in the halophyte Kosteletzkya virginica in response to bombardment with the Particle Inflow Gun
Halophyte Biotechnology Center, College of Marine Studies, University of Delaware Lewes, DE 19958, USA
1To whom correspondence should be addressed. Fax: + 1 302 645 4028. E-mail: jackg@udel. edu
A ‘Particle Inflow Gun’ was constructed to introduce the glucuronidase (GUS) and hygromycin resistance genes into halophytic suspension cells of Kosteletzkya virginica. The transient expression of the GUS gene was associated with the cell culture conditions, physical parameters during the use of the Particle Inflow Gun, and different promoters coupled to GUS. When the CaMV35S promoter was used, the cells adapted at 85 mM NaCl had a similar gene transfer efficiency to those of the non-salt-adapted control, while expression was less in the 170 mM and 255 mM NaCl-adapted cells. Both elevating bombardment pressure to 1.65 mPa and shortening the distance between the cells and the particle holder from 21 cm to 9 cm enhanced GUS expression in the cells grown in four salinity treatments. An ABA-responsive promoter induced the expression of the GUS gene either with 10–4 M ABA or with salts in the post-bombardment medium in both control and NaCl-adapted cell tines. Stable transgenic callus lines were isolated by using hygromycin containing medium after bombarding the suspension cells with the Particle Inflow Gun. The presence of the GUS gene in stable transformants was confirmed not only by histochemical and fluorimetric assays for the GUS activity, but also by Southern hybridization of RT-PCR amplified mANA.
Key words: Transgenic hatophyte, Particle Inflow Gun, bombardment, salt tolerance, transformation