Gene transfer through asymmetric somatic hybrids: Extent and predictability of donor chromosome elimination.

摘要

To produce highly asymmetric somatic hybrids, in order to transfer discrete traits between plant species, donor-chromosome elimination must be better understood. We examined the effects of radiation dose and time in culture on the elimination of donor genetic material. Mesophyll protoplasts of kanamycin-resistant donor Nicotiana plumbaginifolia were gamma-irradiated and fused with recipient mesophyll protoplasts of N. tabacum bearing the sulfur mutation (Su, phenotype yellow). Hybrid calli, selected on kanamycin-medium, were analyzed for N. plumbaginifolia genetic material by either dot-blot hybridization using a N. plumbaginifolia-specific repetitive DNA sequence probe, or by scoring the fraction of green hybrid calli (green = retained donor wild type allele). Both assays showed that the elimination of donor DNA increased with radiation dose, from 5 to 50 krad. At 50 krad, about one third of the hybrids lost 85% or more of the donor genome, which represents the retention of three, or fewer, donor chromosomes. In addition, donor DNA elimination continued for the first 12 months in culture.Alternatively, BrdU (0.1 to 10