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 Agrobaderium-mediated transformation. Transgenic plants were morphologically indistinguishable from wild type and exhibited normal fertility. Canciclovir at lO-5to 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.
Published in Plant Physiology, ed. Donald R. Ort, Volume 104, Issue 3, 1994, pages 1067-1071.
Czakó, M., & Márton, L. (1994). The herpes simplex virus thymidine kinase gene as a conditional negative-selection marker gene in Arabidopsis thaliana. Plant Physiology, 104(3), 1067-1071.
© Plant Physiology, 1994, American Society of Plant Biologists