Running head : Targeting of the Adh 2 gene in rice

The modification of an endogenous gene into a designed sequence by homologous recombination, termed gene targeting, has broad implications for basic and applied research. Rice (Oryza sativa L.), with a sequenced genome of 389 Mb, is one of the most important crops and a model plant for cereals, and the single-copy gene Waxy on chromosome 6 has been modified with a frequency of 1% per surviving callus by gene targeting using a strong positive-negative selection. Since the strategy is independent of gene-specific selection or screening, it is in principle applicable to any gene. However, a gene in the multigene family or a gene carrying repetitive sequences may preclude efficient homologous recombination-promoted gene targeting due to the occurrence of ectopic recombination. Here, we describe an improved gene targeting procedure whereby we obtained nine independent transformed calli having the Adh2 gene for alcohol dehydrogenase 2 modified with a frequency of approximately 2% per surviving callus and subsequently isolated eight fertile transgenic plants without the concomitant occurrence of undesirable ectopic events, even though the rice genome carries four Adh genes, including a newly characterized Adh3 gene, and a copy of highly repetitive retroelements is present adjacent to the Adh2 gene. The results indicate that gene targeting using a strong positive-negative selection can be widely applicable to functional genomics in rice and presumably in other higher plants. www.plantphysiol.org on July 28, 2017 Published by Downloaded from Copyright © 2007 American Society of Plant Biologists. All rights reserved.