Hf. Kaeppler et al., Routine utilization of green fluorescent protein as a visual selectable marker for cereal transformation, IN VITRO-PL, 37(2), 2001, pp. 120-126
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.