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Systematic Reverse Genetics of Transfer-DNA-Tagged Lines of Arabidopsis1
Isolation of Mutations in the Cytochrome P450 Gene Superfamily

Rodney G. Winkler2, Michael R. Frank, David W. Galbraith, René Feyereisen, and Kenneth A. Feldmann*

Department of Plant Sciences (R.G.W., D.W.G., K.A.F.) and Department of Entomology (M.R.F., R.F.), University of Arizona, Tucson, Arizona 85721

We have developed an efficient reverse-genetics protocol that uses expedient pooling and hybridization strategies to identify individual transfer-DNA insertion lines from a collection of 6000 independently transformed lines in as few as 36 polymerase chain reactions. We have used this protocol to systematically isolate Arabidopsis lines containing insertional mutations in individual cytochrome P450 genes. In higher plants P450 genes encode enzymes that perform an exceptionally wide range of functions, including the biosynthesis of primary metabolites necessary for normal growth and development, the biosynthesis of secondary products, and the catabolism of xenobiotics. Despite their importance, progress in assigning enzymatic function to individual P450 gene products has been slow. Here we report the isolation of the first 12 such lines, including one (CYP83B1-1) that displays a runt phenotype (small plants with hooked leaves), and three insertions in abundantly expressed genes. The DNAs used in this study are publicly available and can be used to systematically isolate mutants in Arabidopsis.

1   This work was supported by the National Science Foundation-Department of Energy-U.S. Department of Agriculture (USDA) Joint Program of Collaborative Research in Plant Biology (92-20332), by USDA grant no. 9701472 to K.A.F., R.F., and D.W.G., and by the University of Arizona Agricultural Experiment Station.
2   Present address: Genomica Corp., 4001 Discovery Drive, Suite 130, Boulder, CO 80303.
*   Corresponding author; e-mail feldmann{at}ag.arizona.edu; fax 1-310-825-5275.

Plant Physiol. (1998) 118: 743-750
Copyright Clearance Center:   0032-0889/98/118//08
© 1998 American Society of Plant Physiologists




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