Electrofusion of biochemically well characterized nitrate reductase deficient Nicotiana plumbagnifolia mutants. Studies of optimization and complementation

Abstract

Electrofusion was carried out using biochemically well characterized NR- Nicotiana plumbaginifolia mutants (Cnx 20, Nia 26, NA 36). In analytical experiments, optimal conditions for mesophyll-mesophyll and callus-callus protoplast fusions were assessed. Subsequently, in large scale experiments NR+ somatic hybrids were obtained after mesophyll protoplast fusions between Cnx 20 + Nia 26 as well as after callus protoplast fusions between Cnx 20 + Nia 26 and between Cnx 20 + NA 36. In addition, complementation of the nia mutants Nia 26 and NA 36, each characterized by a distinct biochemical phenotype, was studied using electrofusion. In these experiments no completing NR+ somatic hybrid callus was obtained. As fusion conditions were optimal and fusion products were observed to be formed it was concluded that the nia mutations, although leading to distinct biochemical phenotypes, are allelic. We also studied complementation in short term experiments. NR activity in vivo was assayed 3-4 weeks after fusion. Plants could be regenerated from the majority of the NR+ somatic hybrid calli, resulting from the fusions between Cnx 20 + Nia 26 and Cnx 20 + NA 36. Chromosome numbers of shoot tip cells of glass house grown plants varied between 32-58, the majority having the normal tetraploid number (2n = 40). Most of the plants appeared to be sterile. © 1987.