A fast neutron deletion mutagenesis-based reverse genetics system for plants

A new reverse genetics method has been developed to identify and isolate de letion mutants for targeted plant genes. Deletion mutant libraries are gene rated using fast neutron bombardment. DNA samples extracted from the deleti on libraries are used to screen for deletion mutants by polymerase chain re action (PCR) using specific primers flanking the targeted genes. By adjusti ng PCR conditions to preferentially amplify the deletion alleles, deletion mutants were identified in pools of DNA samples, each pool containing DNA f rom 2592 mutant lines. Deletion mutants were obtained for 84% of targeted l oci from an Arabidopsis population of 51 840 lines. Using a similar approac h, a deletion mutant for a rice gene was identified. Thus we demonstrate th at it is possible to apply this method to plant species other than Arabidop sis. As fast neutron mutagenesis is highly efficient, it is practical to de velop deletion mutant populations with more complete coverage of the genome than obtained with methods based on insertional mutagenesis. Because fast neutron mutagenesis is applicable to all plant genetic systems, this method has the potential to enable reverse genetics for a wide range of plant spe cies.