Resumen:
For the isolation of promoters with specific expression patterns a trapping technology
has been assembled for banana, which allows in planta characterization of candidate
promoters without a priori isolation of the corresponding genes.
Detection and quantitation of luciferase expression was optimized in transgenic banana
for capturing images by an ultrasensitive CCD camera. Time course and stability
studies of luciferase expression were performed to determine time points for early
detection. A promoterless luciferase reporter gene located next to a T-DNA border
region has then been introduced to embryogenic cell suspensions of different banana
cultivars. Screening for luciferase activation has been performed without induction as
well as via various induction treatments during different steps of in vitro regeneration.
Two to three months after transformation, screening of approximately 24,000 transgenic
colonies revealed 16 candidates (0.07%) with constitutive expression. Screening of
12,000 colonies with the SAR (systemic acquired resistance) activator Bion® resulted in
the identification of six (0.05%) inducible candidates. In parallel, out of 774 proliferating
cultures (4-8 months after transformation) four (0.52%) exerted constitutive expression
comparable to the CaMV35S promoter whereas among 157 differentiated in vitro plants
(8-12 months after transformation) activation was observed in eight (5.1%) individuals.
Further screening of colonies, proliferating cultures and in vitro plants has been
performed under temperature, osmotic and aluminium stress conditions as well as after
treatment with paraquat.
Up to now, all candidates have been propagated for plant regeneration and molecular
analysis. Regions upstream to the promoter tags have been cloned by PCR techniques
and sequenced.
