Isolation and characterization of microprotoplasts from APM-treated suspension cells of Nicotiana plumbaginifolia

Subprotoplasts with a DNA content of less than the G1 level (microprotoplasts) were isolated from micronucleated cells of transformed Nicotiana plumbaginifolia ("Doba" line resistant to kanamycin) and characterized cytologically as well as by flow cytometry and Feulgen microdensitometry. Micronuclei were induced upon treatment of the suspension cells with the anti-microtubule drug amiprophos-methyl (APM). Protoplasts were fractionated on a continuous iso-osmotic gradient of Percoll; this resulted in several visible bands. Flow cytometric analysis of fluorescein and nuclear DNA contents after staining with fluorescein and DAPI respectively showed that the main band contained mostly evacuolated, intact (sub)protoplasts. Microprotoplasts contained one or a new micronuclei surrounded by a thin rim of cytoplasm and an intact plasma membrane A maximum of 40% of the microprotoplasts in the fraction just below the main band had a DNA content less than the G1 level, in other fractions this maximum was 20%.