Culture conditions of protoplast-derived cells of nicotiana sylvestris for mutant selection

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Abstract
Large numbers of protoplasts showing reproducible high plating efficiency can be isolated from in vitro propagated, haploid and diploid, plants of Nicotiana sylvestris. Their successful use in the selection of biochemical mutants depends on the establishment of suitable selection parameters: culture medium, cell density, age of cells at selection etc. Plating of protoplasts at low densities as well as simulation and reconstruction experiments of mutant selection were employed to optimize such selection parameters. The results show that some of the principles determined for tobacco protoplast cultures manipulated at low densities or in view of mutant selection are of more general value. However, requirements specific to N. sylvestris protoplast cultures have also been established; they play a decisive part in the successful isolation of resistant mutants in this species.