Isolation and characterisation of nitrate reductase-minus cell-lines of Nicotiana Tabacum

Abstract

The object of this research was to isolate nitrate reductase-minus cell-lines of a higher plant species in order to gain information on the genetic control of nitrate reductase production in higher plants. This demanded a culture system consisting of isolated cells or protoplasts, ideally carrying single-copy genetic information. To this end, haploid Nicotiana plants were raised by another culture and abortive attempts were made to culture haploid sylvestris protoplasts. Callus and cell suspension cultures of haploid N. sylvestris also proved unsuitable but eventually dihaploid M. tabacum suspension cultures were obtained which, it was decided, would be suitable for this work. After treatment of these cultures with EMS, 59 cell-lines were selected for resistance to chlorate, four of these were unable to grow on nitrate medium and were later shown to possess an assembled but Inactive nitrate reductase. None of the foul possessed xanthine dehydrogenase activity indicating that they were molybdenum co-factor (Mo-co) defective lines (cnx variants). These cnx lines are different from other Mo-co defective tabacum lines thus far described and are therefore new types, providing evidence that more than one gene locus is involved in the formation of functional Mo-co in N. tabacum nitrate reductase.