Genetic transformation of bahiagrass (Paspalum notatum Flügge) by visually screening cells expressing green fluorescent protein

Abstract Luminescent proteins, such as the green fluorescent protein (GFP) from the jellyfish Aequorea victoria, have proven to be powerful tools in plant genetic transformation and gene expression studies due to their excellent sensitivity and rapid response. GFP has also the significant advantage that it does not require a substrate, and its expression can be detected in real time in living cells by light excitation. Therefore, GFP has been used as a reliable reporter for plant transformation. In this study, the plasmid modified pBI221 (pBI0809) containing the gfp reporter gene under control of the cauliflower mosaic virus (CaMV) 35S promoter was introduced into mature seed‐derived embryogenic calli by particle bombardment. Sectors containing embryogenic calli with strong GFP fluorescence were identified using a fluorescence viewing system. Cell‐ and tissue‐specific expression of the gfp gene were also investigated in transgenic plants, and stable GFP expression was observed in transgenic calli. As a result of GFP selection, the total time required to produce transgenic calli was reduced by approximately 14 days compared to the time needed when selective agents such as the bar gene are used (50–60 days). Three months after bombardment, regenerated transformants were potted in soil, acclimatized and transferred to the greenhouse. A 480‐bp fragment was amplified and identified as the gfp gene by polymerase chain reaction. The presence of the gfp gene in genomic DNA of three out of nine transgenic bahiagrass lines was confirmed by DNA gel blot hybridization analysis. Transformants showed hybridization patterns suggesting that the gfp gene was present in three copies per regenerant. The GFP expression could be visually detected in all tissues such as leaves, leaf sheaths, anthers and pollen. Overall, the gfp gene can be utilized not only as a reporter gene, but also as a visual selectable marker for bahiagrass transformation.