Sm. De Villiers et al., Biolistic transformation of chincherinchee (Ornithogalum) and regenerationof transgenic plants, PHYSL PLANT, 109(4), 2000, pp. 450-455
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.