Problems associated with the isolation of auxotrophic mutants from plant cell tissue culture.

The problems of auxotrophic mutant selection and preservation in cell tissue culture of Nicotiana sylvestris have been investigated and some progress towards their solution made. Variation in cytological stability between cultures depends on the genotype and culture conditions. Repeated cloning led to a cell line which was more stable than the parental line, although still susceptible to polyploidisation. Comparison within sets of clones showed that mean cell diameter was proportional to mean DNA content, and that the level of dispersion was related to cell size. The more polyploid cells were the least aggregated, and this relationship held even within a culture. The minimum inoculum density in suspension culture was reduced to 100 cells per ml. whereas in agar this was higher, being 3,000 cells per ml. for very small aggregates. Repeated photomicrography allowed the fate of individual cells and aggregates to be followed. The lethality of the mutagen, ethyl methane sulphonate, was determined by vital staining and plating. The suicide enrichment technique employing 5-bromodeoxyuridine 2'deoxyriboside was found to be highly effective in the preferential killing of growing over arrested cells in model culture systems of N. sylvestris and Acer pseudoplatanus. An attempt was made at the isolation of auxotrophic mutants, employing a protocol including mutagenic and enrichment steps. None of the colonies recovered were auxotrophic. Cultures of N. sylvestris are difficult to freeze preserve due to their large cell size and high degree of vacuolation. The best recovery was achieved by pre-growth in mannitol-containing medium, use of glycerol as cryoprotectant, freezing at 1/2°C per minute to -35°C and plunging into liquid nitrogen, followed by rapid thawing. Post-thaw loss of viability upon resuspension, although high, could be reduced by the use of medium iso-osmotic to that in which the cells were frozen