Self-complementary chimeric oligonucleotides (COs) composed of DNA and modified RNA residues were evaluated as a means to (i) create stable, site-specific base substitutions in a nuclear gene and (ii) introduce a frameshift in a nuclear transgene in plant cells. To demonstrate the creation of allele-specific mutations in a member of a gene family, COs were designed to target the codon for Pro-196 of SuRA, a tobacco acetolactate synthase (ALS) gene. An amino acid substitution at Pro-196 of ALS confers a herbicide-resistance phenotype that can be used as a selectable marker in plant cells. COs were designed to contain a 25-nt homology domain comprised of a five-deoxyribonucleotide region (harboring a single base mismatch to the native ALS sequence) f lanked by regions each composed of 10 ribonucleotides. After recovery of herbicide-resistant tobacco cells on selective medium, DNA sequence analyses identified base conversions in the ALS gene at the codon for Pro-196. To demonstrate a site-specific insertion of a single base into a targeted gene, COs were used to restore expression of an inactive green f luorescent protein transgene that had been designed to contain a single base deletion. Recovery of f luorescent cells confirmed the deletion correction. Our results demonstrate the application of a technology to modify individual genetic loci by catalyzing either a base substitution or a base addition to specific nuclear genes; this approach should have great utility in the area of plant functional genomics.Genomics is currently a central component of plant biology research. The gene sequence of Arabidopsis thaliana will soon be available, and many other species are under study. Identification of new genes is occurring at a much faster pace than is the determination of their function. One missing technology in plant biology is the ability to selectively and reliably create site-specific ''gene knockouts'' or homologous recombination of genes of interest or of unknown function. The phenomena of gene silencing (e.g., antisense and cosuppression) provides a method for understanding gene function through the creation of transgenic crops expressing a gene sequence that silences the endogenous gene. However, this approach is not suitable for functional genomic studies of individual members of multigene families or genes with similar sequence, and is sometimes problematic in other technical aspects (1).A technology currently being explored in prokaryotic and eukaryotic systems uses self-complementary chimeric oligonucleotides (COs) comprised of DNA and 2Ј-O-methyl RNA to target and mutate genes in vivo. These COs are designed to have one or more bases that do not pair with the endogenous gene sequence. This approach was successfully used to modify endogenous genes of mammalian cells (2-5) in a site-specific and genetically inheritable manner. Recently, Alexeev and Yoon (5) have demonstrated that permanent in vivo conversions result in phenotypic changes in mouse melanocytes. It has been hypothesized that the mechan...