Isolation, culture, and fusion of tobacco and potato protoplasts
Isolation of plant protoplasts, followed by fusion and subsequent regeneration of somatic hybrids, are remarkable techniques that allow exchange of genetic information between sexually incompatible species. Success in application of these techniques was first achieved with tobacco for which Nagata and Takebe developed a protocol for cell wall regrowth and cell division, followed eventually by plant regeneration. This chapter illustrates the basic procedures of protoplast isolation (PI) and fusion. Tobacco is susceptible to infestation with whitefly under greenhouse conditions, so treatment with a systemic insecticide shortly after transplanting is recommended. The best source of plant material for protoplast manipulation of potato is in vitro plantlets. In vitro potato can harbor bacterial contaminants that are not apparent during routine subculture on basal medium. For tobacco PI, five media are needed. Three of these are simple variations of PI medium and can be made from the same preparation of PI.