ELECTROPORATION-MEDIATED AND SILICON-CARBIDE FIBER-MEDIATED DNA DELIVERY IN AGROSTIS-ALBA L (REDTOP)

The results of DNA delivery experiments in Agrostis alba by electroporation into embryogenic suspension culture-derived protoplasts and by the silicon carbide fiber-mediated method into intact suspension cells are described, using the two plasmid constructs. pFF19G and wheat dwarf virus (WDV)-derived pWI-GUS. The beta-glucuronidase (GUS) activity of electroporated protoplasts was detected fluorometrically. The optimal electrical conditions for DNA delivery into the protoplasts was estimated to be around 750 V/cm for 38 ms. When electroporated protoplasts were incubated for a prolonged period or time, the GUS expression of pWI-GUS treated cells increased progressively during 5 days after electroporation, indicating the replication of this vector in the cells. The GUS expression in the cells treated with silicon carbide fibers and plasmid DNA was detected histochemically. The frequency of GUS expression units tended to be highest when DNA was delivered to the cells on the 6th day after subculture, giving an estimated frequency of GUS-expressed cell colonies of about 1%.