AGROBACTERIUM-MEDIATED TRANSFORMATION OF SUBTERRANEAN CLOVER (TRIFOLIUM-SUBTERRANEUM L)

We have developed a rapid and reproducible transformation system for s ubterranean clover (Trifolium subterraneum L.) using Agrobacterium tum efaciens-mediated gene delivery. Hypocotyl segments from seeds that ha d been allowed to imbibe were used as explants, and regeneration was a chieved via organogenesis. Glucose and acetosyringone were required in the co-cultivation medium for efficient gene transfer. DNA constructs containing four genes encoding the enzymes phosphinothricin acetyl tr ansferase, beta-glucuronidase (GUS), neomycin phosphotransferase, and an alpha-amylase inhibitor were used to transform subterranean clover. Transgenic shoots were selected on a medium containing 50 mg/L of pho sphinothricin. Four commercial cultivars of subterranean clover (repre senting all three subspecies) have been successfully transformed. Sout hern analysis revealed the integration of T-DNA into the subterranean clover genome. The expression of the introduced genes has been confirm ed by enzyme assays and northern blot analyses. Transformed plants gro wn in the glasshouse showed resistance to the herbicide Baste at appli cations equal to or higher than rates recommended for killing subterra nean clover in field conditions. In plants grown from the selfed seeds of the primary transformants, the newly acquired gene encoding GUS se gregated as a dominant Mendelian trait.