A rapid assay for genetic complementation of nitrate reductase deficiency via bulk protoplast fusion

Abstract

Genetic complementation of different nitrate reductase-deficient (NR-) Nicotiana plumbaginifolia cell lines could be recognized in the described fusion disc technique as early as 5–10 days after protoplast fusion. Protoplasts of previously characterized mutant cell lines, belonging to four different complementation groups, were mixed in pairwise combinations and fused by the drop-wise fusion technique at very high protoplast densities (106 protoplasts in an 8–10 mm spot) which resulted in the formation of a ‘fusion disc’ on the bottom of the petri dish. After 5–10 days of incubation in K3 medium the restoration of NR activity could be detected directly in the original culture by the in vivo NR assay. Such a rapid test giving information about the genetic complementation of different auxotrophic cell lines has not previously been published for plants.