Somatic hybridisation using a double mutant of Nicotiana tabacum

Abstract

Leaf mesophyll protoplasts of a nitrate reductase deficient, streptomycin resistant double mutant of Nicotiana tabacum were fused with cell suspension protoplasts of wild type N. rustica. Hybrid colonies were selected for nitrate reductase proficiency and streptomycin resistance. Green, actively proliferating colonies were recovered in the selection medium. Eleven of twenty green colonies, transferred to regeneration medium, produced plants. Regenerated plants were analysed for their vegetative and floral characteristics, Fraction 1 protein polypeptide composition and leaf esterases. All the eleven regenerants were nuclear somatic hybrids possessing the chloroplast of the N. tabacum parent. By developing a double mutant with a negative (auxotrophic) and a positive (resistance) selection marker in a species of interest, this species can be hybridised by somatic cell fusion with wild type species lacking selectable markers.