Somatic hybridization in Nicotiana: Restoration of photoautotrophy to an albino mutant with defective plastids

Abstract

Protoplasts of a cytoplasmic albino mutant of Nicotiana tabacum L. characterized by a deficient chloroplast genome were fused with protoplasts of a nitrate-reductase deficient mutant (NR(-)) of N. tabacum. Somatic hybrids were obtained where the genome of the NR(-) mutant was complemented by the cytoplasmic albino mutant which could synthesize an active nitrate reductase, and the chlorophyll deficiency in the albino mutant was restored by the chloroplasts from the NR(-) mutant. Cybrids were also obtained in which the deficient plastids of the cytoplasmic albino mutant were replaced by normal chloroplasts from the NR(-) mutant. The system used permitted a simple selection of the hybrids and the cybrids. The NR(-) mutant was excluded at the cellular level by transfer of the cells to medium deficient in reduced nitrogen. The cytoplasmic albino mutant grew well on the selective nitrate medium. However, during callus formation, clear differences in the morphology and pigmentation of the calli were found which permitted selection for photoautotrophy at the callus level. The hybrid or cybrid nature of the plants was confirmed by examination of their morphology and chromosome number. Although the fusion partners come from the same species, only one plant showed the white-green variegated pattern typical of that of the cytoplasmic albino parent, indicating that segregation of plastids occurred during development of the calli and regeneration of the plants.