Efficient induction and selection of chloroplast-encoded antibiotic-resistant mutants in Nicotiana

Abstract

A high rate of plastome-encoded mutations was induced in Nicotiana by exposing seeds to N-nitroso-N-methylurea. Seeds then were subjected to nutritional and in vitro selection procedures for systematic isolation of plastome-dependent antibiotic-resistant plants. Multiple flowering lines resistant to streptomycin, spectinomycin, lincomycin, or chloramphenicol were obtained. A detailed analysis of the streptomycin-resistant lines is presented. Sexual hybridization, cybrid formation following protoplast fusion, and in organello protein synthesis were used to rigorously assign the mutations to the chloroplast genome. The efficient rates of mutagenesis combined with the in vitro mass-screening procedures described here should facilitate investigation of fundamental aspects of chloroplast genetics in higher plants.