Regeneration of fully nitrate reductase — deficient mutants from protoplast culture of Nicotiana plumbaginifolia (Viviani)

Abstract

Protoplast-derived colonies of haploid N. plumbaginifolia were selected for by chlorate resistance in media supplemented with casamino acids. Eighty resistant lines were confirmed by a second passage on a higher concentration of chlorate. Frequency of spontaneous mutation ranged from 10(-5) to 10(-6). Fifty of the resistant lines could be regenerated into plants, and 30 were characterized biochemically. Ninety percent were fully deficient for nitrate reductase activity. The lines were further tested for xanthine dehydrogenase activity and subsequently classified as defective in the apoenzyme (nia type, 26 lines) or the cofactor (cnx type, 4 lines). Two groups had been identified up until now within the cnx type by growth tests on high concentrations of molybdate supplied to the medium. Nitrate reductase deficiency was stably and continously expressed in both variant cell cultures and regenerants. Genetic analysis demonstrated that nitrate reductase deficiency was inherited as a single recessive nuclear gene.