A high rate of plastome-encoded mutations was induced in Nicotiana by exposing seeds to N-nitroso-Nmethylurea. Seeds then were subjected to nutritional and in vitro selection procedures for systematic isolation of plastomedependent 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.An extensive body of literature, dating back more than half a century, exists on chloroplast inheritance in higher plants (1). The use of nuclear genes capable of increasing plastome (i.e., chloroplast) mutation rates (2) and studies with exceptional biparentally inherited plastids (3) continue to contribute to our knowledge of organelle inheritance. Likewise, recent advances in molecular analysis of the chloroplast genome (4) and the development of protoplast-fusion methodologies for plants (5) have paved the way to a more thorough understanding of plastome genetics and nuclear-genome/plastome interactions. Nevertheless, hypotheses about the rules governing chloroplast genetics usually are the result of extrapolation from the alga Chlamydomonas rather than based on intrinsic advancements with higher plants (1).Spontaneous plastome-dependent variegations in plants occur at a frequency of about 5 x 10-4; however, the spontaneous mutation rate for specific traits is estimated to be less than 10-9 (2). Consequently, successful isolation of specific mutants probably requires a plastome-targeted mutagenic agent and a selection scheme that allows sorting-out of the multicopy plastome in the somatic dividing cells. Mutagenesis followed by selection for specific plastome-encoded traits in callus culture has been studied in the genus Nicotiana. The results to date are one lincomycin-(6) and two streptomycin-resistant (7,8) cell lines. In this report we evaluate the chemical mutagen N-nitroso-N-methylurea (NMU), which efficiently causes plastome lesions (9), and cell culture and nutritional procedures that potentiate mutant expression during plastid segregation in the developing plant. The combined application of these procedures has resulted in the isolation of numerous Nicotiana mutant cell lines resistant to streptomycin, spectinomycin, lincomycin, or chloramphenicol.
MATERIALS AND METHODSPlant Material. Nicotiana tabacum cultivar (cv.) Xanthi is a self-fertile line. N. tabacum line 92 is a cytoplasmic malesterile, alloplasmic substitution line containing N. undulatatype cytoplasm (10). Clonal line VBW, containing albino plastids (10), is from a variegated N. tabacum mutant, Dpl, containing defective plastids...