M. Czako et L. Marton, THE HERPES-SIMPLEX VIRUS THYMIDINE KINASE GENE AS A CONDITIONAL NEGATIVE-SELECTION MARKER GENE IN ARABIDOPSIS-THALIANA, Plant physiology, 104(3), 1994, pp. 1067-1071
The human herpes simplex virus thymidine kinase type 1 gene (HSVtk) ac
ts as a conditional lethal marker in mammalian cells. The HSVtk-encode
d enzyme is able to phosphorylate certain nucleoside analogs (e.g. gan
ciclovir, an antiherpetic drug), thus converting them to toxic DNA rep
lication inhibitors. The utility of HSVtk as a conditional negative-se
lection marker was explored in Arabidopsis thaliana (L.) Heynh. HSVtk
was introduced into Ara bidopsis by Agrobacterium-mediated transformat
ion. Transgenic plants were morphologically indistinguishable from wil
d 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 af
fect the wild-type cultures. There was a 35-fold reduction in shoot re
generation 8 d after transfer to shoot-induction medium. Negative sele
ction against HSVtk activity along with kanamycin selection was also e
fficient in Agrobacterium-mediated gene transfer experiments. Shoot re
generation was 25 times lower on double-selective (ganciclovir plus ka
namycin) 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.