PLANT PHYSIOLOGY , Vol 104, Issue 3 1067-1071, Copyright © 1994 by American Society of Plant Biologists
The Herpes Simplex Virus Thymidine Kinase Gene as a Conditional Negative-Selection Marker Gene in Arabidopsis thaliana
M. Czako and L. Marton
Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208
The human herpes simplex virus thymidine kinase type 1 gene (HSVtk) acts as
a conditional lethal marker in mammalian cells. The HSVtk-encoded enzyme is
able to phosphorylate certain nucleoside analogs (e.g. ganciclovir, an
antiherpetic drug), thus converting them to toxic DNA replication
inhibitors. The utility of HSVtk as a conditional negative-selection marker
was explored in Arabidopsis thaliana (L.) Heynh. HSVtk was introduced into
Arabidopsis by Agrobacterium-mediated transformation. Transgenic plants
were morphologically indistinguishable from wild type and exhibited normal
fertility. Ganciclovir at 10-5 to 10-4 M drastically reduced shoot
regeneration on transgenic, HSVtk+ root explants or callus formation on
HSVtk+ leaf explants but did not affect the wild-type cultures. There was a
35-fold reduction in shoot regeneration 8 d after transfer to
shoot-induction medium. Negative selection against HSVtk activity along
with kanamycin selection was also efficient in Agrobacterium-mediated gene
transfer experiments. Shoot regeneration was 25 times lower on
double-selective (ganciclovir plus kanamycin) plates than in the kanamycin
control. This regeneration rate in double-selective plates is in the range
of the frequency of shoots normally escaping kanamycin selection in
Arabidopsis cultures.
