Ar. Carlson et al., Visual screening of microspore-derived transgenic barley (Hordeum vulgare L.) with green-fluorescent protein, PL CELL REP, 20(4), 2001, pp. 331-337
The green-fluorescent protein (GFP) gene from the Pacific Northwest jellyfi
sh, Aequorea victoria, was used as a screenable marker in the production of
transgenic barley plants. Isolated barley microspore culture was biolistic
ally transformed with two synthetic forms of GFP, sgfp and pgfp. Thirty-sev
en fluorescing multicellular structures were isolated using epifluorescent
microscopy. Sixteen structures developed shoots, but only five regenerated
into green plants. Three events had been co-bombarded with P-glucuronidase
(gus) and assayed positive for gus expression in the leaves, and all five e
vents were positive for gfp expression. The expected transgene band size wa
s PCR-amplified from all five plants, and Southern blots performed on three
plants revealed unique patterns of gfp transgene integration. Fluorescent
in situ hybridization also revealed the transgenic status and hemizygous na
ture of all the events. GFP-based visual screening provides a viable altern
ative method to chemical selection of transgenic plants from barley microsp
ore culture.