Present address: Cropsolution, Inc., PO Box 14069, Research Triangle Park, NC 27709–4069, USA.
Gene targeting in Arabidopsis
Article first published online: 11 JAN 2002
The Plant Journal
Volume 28, Issue 6, pages 671–677, December 2001
How to Cite
Hanin, M., Volrath, S., Bogucki, A., Briker, M., Ward, E. and Paszkowski, J. (2001), Gene targeting in Arabidopsis. The Plant Journal, 28: 671–677. doi: 10.1046/j.1365-313x.2001.01183.x
- Issue published online: 11 JAN 2002
- Article first published online: 11 JAN 2002
- Received 22 June 2001; revised 10 September 2001; accepted 12 September 2001.
- homologous recombination;
- gene targeting;
- protoporphyrinogen oxidase (PPO)
Precise modification by gene targeting (GT) provides an important tool for studies of gene function in vivo. Although routine with many organisms, only isolated examples of GT events have been reported for flowering plants. These were at low frequencies precluding reliable estimation of targeting efficiency and evaluation of GT mechanisms. Here we present an unambiguous and straightforward system for detection of GT events in Arabidopsis using an endogenous nuclear gene encoding protoporphyrinogen oxidase (PPO), involved in chlorophyll and heme syntheses. Inhibition of PPO by the herbicide Butafenacil results in rapid plant death. However, the combination of two particular mutations renders PPO highly resistant to Butafenacil. We exploited this feature for selection of GT events by introducing the mutations into the PPO gene by homologous recombination. We have estimated the basal GT frequency to be 2.4 × 10−3. Approximately one-third of events were true GT (TGT) leading to the anticipated modification of the chromosomal PPO copy. The remaining events could be classified as ectopic GT (EGT) arising by modification of vector DNA by the chromosomal template and its random integration into the Arabidopsis genome. Thus the TGT frequency in our experimental setup is 0.72 × 10−3. In view of the high efficiency of Arabidopsis transformation, GT experiments of a reasonable size followed by a PCR screen for GT events should also allow for modification of non-selectable targets. Moreover, the system presented here should contribute significantly to future improvement of GT technology in plants.