Abstract
Defined cybridization was performed by one-to-one electrofusion (microfusion) of preselected protoplast-cytoplast pairs of male-fertile, streptomycin-resistant Nicotiana tabacum and cytoplasmic male-sterile, streptomycin-sensitive N. tabacum cms (N. bigelovii), followed by microculture of the fusion products until plant regeneration. Dominant selectable markers, namely, kanamycin resistance (nptII) and hygromycin B resistance (hpt) genes had been previously integrated in the nuclear genomes of the otherwise almost fully isogenic parental strains using direct gene transfer to protoplasts. In addition to chromosome counts indicating the expected allotetraploid tobacco count of 48, the absence of the nucleus from the cytoplast donor line was confirmed by Southern blot hybridization using nptII and hpt probes, as well as by an in vitro selection test with leaf expiants and the corresponding enzyme assays for 30 cybrids. The cytoplasmic composition of the cybrids obtained was analyzed for chloroplast type using the streptomycin resistance/sensitivity locus. The fate of mitochondria in cybrids was checked by species-specific patterns in Southern analysis of restriction endonuclease digests of total DNA with N. sylvestris mitochondrial DNA probes.
Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
