BIOLISTIC TRANSFER OF LARGE DNA FRAGMENTS TO TOBACCO CELLS USING YACSRETROFITTED FOR PLANT TRANSFORMATION

To determine whether large DNA molecules could be transferred and inte grated intact into the genome of plant cells, we bombarded tobacco sus pension cells with yeast DNA containing artificial chromosomes (YACs) having sizes of 80, 150, 210, or 550 kilobases (kb). Plant selectable markers were retrofitted on both YAC arms so that recovery of each arm in transgenic calli could be monitored. Stably transformed calli resi stant to kanamycin (300 mg/L) were recovered for each size of YAC test ed. Two Of 12 kanamycin-resistant transformants for the 80 kb YAC and 8 of 29 kanamycin-resistant transformants for the 150 kb YAC also cont ained a functional hygromycin gene derived from the opposite YAC arm. Southern analyses using probes that spanned the entire 55 kb insert re gion of the 80 kb YAC confirmed that one of the two double-resistant l ines had integrated a fully intact single copy of the YAC DNA while th e other contained a major portion of the insert. Transgenic lines that contained only one selectable marker gene from the 80 kb YAC incorpor ated relatively small portions of the YAC insert DNA distal to the sel ectable marker. Our data suggest genomic DNA cloned in artificial chro mosomes up to 150 kb in size have a reasonable likelihood of being tra nsferred by biolistic methods and integrated intact into the genome of plant cells. Biolistic transfer of YAC DNA may accelerate the isolati on of agronomically useful plant genes using map-based cloning strateg ies.