THE HERPES-SIMPLEX VIRUS THYMIDINE KINASE GENE AS A CONDITIONAL NEGATIVE-SELECTION MARKER GENE IN ARABIDOPSIS-THALIANA

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.