Large scale isolation of maternally inherited lincomycin resistance mutations, in diploid Nicotiana plumbaginifolia protoplast cultures

Summary Lincomycin-resistant clones were isolated in diploid protoplast cultures of Nicotiana plumbaginifolia. Selection of the resistant clones was based on the ability of resistant calli to green in the presence of the antibiotic (1,000 mg l^-1). Sensitive colonies formed white calli under the same conditions. In the absence of mutagenic treatment the frequency of the resistant clones was 1.0×10^-4. This frequency could be increased up to 5.8×10^-4 and 7.2×10^-4 by treatment with 0.1 mM and 0.3 mM N-ethyl-N-nitrosourea (NEU), respectively.Regenerated plants of 56 clones were tested for lincomycin resistance. Regenerates from all but seven clones were resistant to lincomycin, as demonstrated by leaf assay. The lincomycin-resistant regenerates tested were also resistant to clindamycin (a lincomycin derivative), but sensitive to streptomycin.Regenerated plants in 17 clones were fully fertile and inherited lincomycin resistance maternally. Segregation for lincomycin resistance was observed in the seed progeny of five clones, which indicated maintenance of mixed cytoplasmic determinants after plant regeneration. Seed transmission of lincomycin resistance was confirmed in an additional 17 clones but the mode of inheritance (maternal or Mendelian) was not determined because of pollen sterility or reduced seed germination ability. These defects first appeared when the higher concentration of NEU was used. Various pigment deficiencies were also observed in a few clones.