Visual selection of transgenic barley (Hordeum vulgare L.) structures and their regeneration into green plants
The production of transgenic plants is providing plant breeding with new tools to transfer genetic traits across species barriers. A novel approach to selecting transgenic material is visual screening using Aequoreo victoria's green fluorescent protein (GFP). My hypothesis was that visual screening would improve the efficiency of selecting transgenic barley (Hordeum vulgare L.) plants. A number of GFP plasmids were tested for transient expression. Barley microspore cultures from cultivars Igri, Trinity and Manley were bombarded with gold particles carring either GFP, or a combination bombardment of GFP and Beta-Glucuronidase (GUS) expression vectors. An optimization experiment for bombardment parameters using GFP transient expression was performed and revealed that the combination of 0.6 micron gold particle size and a target distance of 6 cm yielded the highest transient expression. Thirty-eight GFP expressing multicellular structures were isolated from bombarded microspore cultures and seven regenerated into green plants. The genomic DNA from six surviving plants was analyzed by polymerase chain reaction (PCR) and found to be positive for the introduced marker genes. The three plants from co-bombardment also tested positive for GUS expression in leaves. Fluorescent in-situ hybridization (FISH) confirmed that one of the putative transgenic plants was transformed. Cytological examination of mitotic root tip cells from the transgenic plants determined that five plants were diploid (doubled haploids) and one was an autotetraploid. This visual identification technique allowed the transgenic multicellular structures to be identified and removed from the initial culture plate, reducing the time and labour involved in transgenic selection.