M2 seed screening for nitrate reductase deficiency in Nicotiana plumbaginifolia

Abstract

Nitrate reductase-deficient mutants can be isolated from protoplast-derived cells on the basis of chlorate resistance. Since nitrate assimilation is a highly regulated and tissue specific process, it was expected that mutant selection at the plant level could lead to the identification of new genes involved in the control of nitrate assimilation. Three selective procedures were compared for the selection of mutants impaired in nitrate assimilation, from M2 seeds: chlorate selection, and rescue of plants specifically unable to grow on low, or high, nitrate concentrations. The rescue of some of the plantlets unable to grow on nitrate was achieved through a shoot culture step by treatment with growth regulators. All three procedures led to comparable frequencies of around 5 × 10 −3 in the isolation of mutants totally impaired for nitrate assimilation. The majority of these mutants were of the cnx type, as opposed to mutants selected for chlorate resistance at the cell level, which are predominantly of the nia type. nia mutants and mutants with leaky phenotypes were also identified, but unexpectedly no other class of mutants totally impaired in nitrate assimilation was identified among isolated mutants. The implications of these data are that M2 seed screening can be used as an alternative route to mutant selection at the cell level for the isolation of mutants totally impaired in nitrate assimilation. This approach can be envisaged for the isolation of nitrate reductase-deficient mutants from species recalcitrant to in vitro manipulation at the cell level and subsequent regeneration into fertile plants.