Meetings of the Royal Botanical Society of The Netherlands

Study of organelle segregation and recombination in fusion products requires the use of selectable, cytoplasmatically encoded mutations. Recently, Avive Galum (Mol. Gen. Genet. 1988,215:128 133) was able to isolate a mitochondrially encoded oligomycinresistant mutant of Nicotiana sylvestris. The antibiotic compound oligomycin binds to the membrane of the mitochondrial ATPase and inhibits the proton translocation. Genetic and molecular analyses of the oligomycin-resistant mutants in Saccharomyces cerevisiae showed that they carry nucleotide changes in the mitochondrial genes coding subunit 6 or 9 of the mitochondrial ATPase. A procedure for the isolation of oligomycinresistant micro-calli in Petunia has been developed. Protoplasts of different Petunia species were isolated in a medium with CPW-salts (Frearson, E.M. el al. Dev. Biol. 1973, 33: 130-137) 0-3 m sucrose, 100mg/l 2-(.V-morpholino)ethane sulphonic acid (MES), 1% cellulase and 0-2% macerozyme. The protoplasts were washed in the same medium without enzymes and plated in culture medium TM2GZ with NAA (1 mg/1) and zeatin (I mg/1). After 2 weeks of culturing, the dividing protoplasts were mixed with an equal amount of 0-6% Seaplaque agarose in TM2GZ. Oligomycin (0-1-5 mg/1) was added to the medium at different times during culture. The best results were found after 2 weeks of culturing. The addition of oligomycin significantly decreased the amount of calli growing. Having assessed the parameters for selection several experiments were performed. From each experiment independent calli were picked and recultured on medium with 1 -5 mg/1 oligomycin. Generally, calli isolated from plates with a low concentration of oligomycin did not grow as fast as those isolated from plates with a higher concentration of oligomycin. A number of Petunia species gave regeneration. The plants need to be checked for oligomycin resistance. As selection for oligomycin resistance at plant level seems impossible, protoplasts must be isolated from the oligomycin-resistant plants to determine whether they are still oligomycin resistant.