Biolistic transformation of chincherinchee (Ornithogalum) and regeneration of transgenic plants

Ornithogalum (chincherinchee) is a genus in the Hyacinthaceae. It is popular as a cut flower or pot plant. However, susceptibility to disease, especially ornithogalum mosaic virus, prevents commercial exploitation of micropropagated hybrids. Provided that it is possible to transform Ornithogalum, this problem might be alleviated by the transfer of genes that code for resistance to the virus. The purpose of this study was to develop a transformation protocol using the pat gene as a selectable marker. Callus, induced on leaf segments of an Ornithogalum thyrsoides×O. dubium hybrid cultured in vitro, was bombarded with a particle gun 4–6 weeks after initiation. We first used β-glucuronidase transient expression to optimise the bombardment parameters and then for stable transformation used both a conventional microprojectile-mediated method as well as a modification that entailed complexing single-stranded p35SAC DNA, containing the pat gene, with histone H1 prior to bombardment. Transgenic plants were regenerated from the bombarded tissues and cultured on a medium containing 15 μM phosphinothricin as selective agent. Rooted plants were tested for the presence of the pat gene by polymerase chain reaction. Integration of the gene into the genomic DNA was verified by Southern blotting. Northern blots, enzyme-linked immunosorbent assay, and leaf paint assays with the herbicide Ignite® (glufosinate ammonium) confirmed expression of phosphinothricin acetyl transferase, the enzyme that detoxifies the herbicide.