Routine utilization of green fluorescent protein as a visual selectable marker for cereal transformation

Development of new selectable markers is needed to increase the efficiency and flexibility of plant transformation, and to overcome drawbacks sometime s associated with use of existing markers. A useful alternative to chemical -based selection systems would be a system using visual screening to obtain transgenic lines. Investigations were carried out to determine if the gree n fluorescent protein (gfp) gene could be utilized alone as a visual screen able marker to produce stably transformed, fertile oat plants. Twelve exper iments were conducted in which gfp-based selection was utilized to obtain r outinely stable transgenic lines in oat. A synthetic gfp gene under the con trol of the maize ubiquitin promoter was delivered into embryogenic oat cal lus via microprojectile bombardment. Cell clusters (1-3 mm) expressing gfp were visually identified using epifluorescence, microscopy and physically i solated approximately 3 wk post-bombardment. Fertile, gfp-expressing T0 pla nts were regenerated from 78% of the glowing cell sectors placed on regener ation medium. T0 plants from 55% of the events produced gfp-expressing prog eny in a 3:1 Mendelian ratio. Southern blot and PCR analysis confirmed tran sgene integration and transmission to progeny. Expression of gfp did not re duce plant growth or fertility. Transgene copy number and integration patte rns were similar to those in transgenic plants derived from chemical-based selection systems. The mean transformation frequency, based on fertile even ts obtained per bombarded plate, was 1.8%. Over 180 independent transgenic oat lines were produced, to date, using only visual screening for expressio n of gfp, demonstrating efficiency and repeatability of the selection syste m.