SELECTION OF T-DNA-TAGGED MALE AND FEMALE GAMETOPHYTIC MUTANTS BY SEGREGATION DISTORTION IN ARABIDOPSIS

As a strategy for the identification of T-DNA-tagged gametophytic muta nts, we have used T-DNA insertional mutagenesis based on screening for distorted segregation ratios by antibiotic selection. Screening of si milar to 1000 transgenic Arabidopsis families led to the isolation of eight lines showing reproducible segregation ratios of similar to 1:1, suggesting that these lines are putative gametophytic mutants caused by T-DNA insertion at a single locus. Genetic analysis of T-DNA transm ission through reciprocal backcrosses with wild type showed severe red uctions in genetic transmission of the T-DNA through the male and/or f emale gametes. Direct evidence for mutant phenotypes in these lines wa s investigated by DAPI staining of mature pollen grains and by the ana lysis of seed set and embryo sac morphology in deal-ed ovules. One lin e, termed limped pollen, showed a novel pollen phenotype in that the g enerative cell failed to migrate inward after pollen mitosis I, such t hat the generative or sperm cells remained against the pollen wall. Tw o other lines, andarta and tistrya, were defective in female transmiss ion and showed an early arrest of embryo sac development with the viab le megaspore not initiating the nuclear division cycles. These data de monstrate the efficacy of a segregation ratio distortion strategy for the identification of T-DNA-tagged gametophytic mutants in Arabidopsis .