Transgenic plants of colonial bentgrass from embryogenic callus via Agrobacterium-mediated transformation

Colonial bentgrass (Agrostis tenuis Sibth. Fl. Oxen.) is a cool-season turfgrass used on fairways in golf courses. The object of this study was to develop a more efficient, reliable and repeatable approach in transforming the grass using Agrobacterium ( strain LBA4404), in which beta-glucuronidase (gus) gene was used as a reporter and hygromycin phosphotransferase (hpt) gene as a selectable marker. This vector was effective in transforming 7-week-old calluses derived from mature seeds cultured on MS medium supplemented with 2,4-D. A two-step solid medium selection with increasing hygromycin concentration (from 50 to 70 mg l(-1)) was used to obtain resistant calluses. Hundreds of transgenic plants have been produced from several independent transformed calluses. The presence of functional beta-glucuronidase (GUS) was detected in hygromycin-resistant calluses, young leaves and roots of transgenic plants. The transgenic plants collected from greenhouse showed strong resistance to 50 mg l(-1) hygromycin solution. Four putative transgenic plants and one control plant were randomly chosen and analyzed by Southern blot analysis. Bands corresponding to the hpt gene were clearly shown in transgenic plants.