Biolistic transformation of chincherinchee (Ornithogalum) and regenerationof transgenic plants

Ornithogalum (chincherinchee) is a genus in the Hyacinthaceae. It is popula r as a cut flower or pot plant. However, susceptibility to disease, especia lly ornithogalum mosaic virus, prevents commercial exploitation of micropro pagated hybrids. Provided that it is possible to transform Ornithogalum, th is problem might be alleviated by the transfer of genes that code for resis tance to the virus, The purpose of this study was to develop a transformati on protocol using the pat gene as a selectable marker, Callus, induced on l eaf segments of an Ornithogalum thyrsosides x O. dubium hybrid cultured in vitro, was bombarded with a particle gun 4-6 weeks after initiation. We fir st used beta-glucuronidase transient expression to optimise the bombardment parameters and then for stable transformation used both a conventional mic roprojectile-mediated method as well as a modification that entailed comple xing single-stranded p35SAC DNA, containing the pat gene, with histone Ht p rior to bombardment. Transgenic plants were regenerated from the bombarded tissues and cultured on a medium containing 15 mu M phosphinothricin as sel ective agent. Rooted plants were tested for the presence of the pat gene by polymerase chain reaction. integration of the gene into the genomic DNA wa s verified by Southern blotting. Northern blots, enzyme-linked immunosorben t assay, and leaf paint assays with the herbicide Ignite(R) (glufosinate am monium) confirmed expression of phosphinothricin acetyl transferase, the en zyme that detoxifies the herbicide.