Visual screening of microspore-derived transgenic barley (Hordeum vulgare L.) with green-fluorescent protein

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.