MICROPROTOPLAST MEDIATED TRANSFER OF SINGLE SPECIFIC CHROMOSOMES BETWEEN SEXUALLY INCOMPATIBLE PLANTS

Microprotoplast-mediated chromosome transfer (MMCT) through fusion of small (subdiploid) microprotoplasts of a transgenic triploid potato (S olanum tuberosum) cell line with leaf protoplasts of tobacco (Nicotian a tabacum) and the wild tomato species Lycopersicon peruvianum is repo rted. The microprotoplasts contained one or a few chromosomes. Monosom ic addition plants were produced from the fusion products. We employed mass-scale induction of micronuclei in donor suspension cells of pota to using the microtubule inhibitor Cremart. Protoplasts were isolated from micronucleated cells after incubation in a cell wall digesting en zyme mixture. The microprotoplasts were isolated from the micronucleat ed protoplasts by high-speed centrifugation. By using sequential filtr ation, small microprotoplasts containing one or few chromosomes were s eparated from the bigger subdiploid microprotoplasts. These small micr oprotoplasts were fused with recipient protoplasts of tobacco or tomat o using polyethylene glycol. The selectable marker kanamycin resistanc e (Kan(R)) and the reporter gene beta-glucuronidase (gus), carried by the donor potato chromosome, were used for the selection of fusion pro ducts and the isolation of hybrid calli. Several monosomic addition pl ants were obtained within the short period of 3-4 months after fusion. These contained one potato chromosome carrying a single copy of gus a nd one or two copies of the neomycin phosphotransferase (nptII) gene c onferring Kan(R), and the complete set of chromosomes of tobacco or to mato, as revealed by genomic in situ hybridization and Southern blot h ybridization. The alien genes, gus and nptII, were stably expressed in both the tobacco and tomato backgrounds. They were transmitted to the progeny after backcrossing to tomato. Monosomic and disomic additions , and some introgression plants showing integration of gus and nptII i n the tomato genome, were recovered in the first backcross progeny. Th e potential value of MMCT for the transfer of economically important t raits, genome analysis, and gene expression is discussed.