Abstract:
We have developed a fast, sensitive and high-throughput method for the identification and isolation in
bananas of novel promoters and genes. Following the Agrobacterium-mediated transformation of
embryogenic cell suspension cultures with a promoterless luciferase (luc) gene, candidate promoters are
characterized in planta during the early stages of in vitro development. Screening for luciferase (LUC)
enzymatic activity in transgenic cell colonies has been performed without induction treatments. We have
previously reported that transformation of a luciferase reporter trap linked to the left T-DNA border resulted
in an activation frequency of 0.03-0.07%.
All tagged candidates have now been propagated for plant regeneration and molecular analysis. Southern
hybridization and sequence analysis of several independent lines demonstrated the integration of 2-6 T-DNA
copies in the genome with the frequent presence of the vector backbone or complex
rearrangements. Therefore, TAIL-PCR products from selected tagged lines were individually cloned and
sequenced.
One tagged line displayed constitutive expression higher than that of the CaMV 35S promoter, which
persisted in the regenerated plants, both in vitro and in the greenhouse. Detailed analysis of the LUC
expression in all plant tissues will be presented. One of the T-DNA insertions in this line occurred in a
metallothionein-like protein gene, demonstrating the feasibility of the technique with bananas.
An improved tagging construct, which contains the codon-optimized luc+ gene fused close to the right T-DNA
border, has been prepared. Transformation with this construct resulted in a 40-fold increase in
activation frequency (up to 2.5% without induction) compared to the original tagging construct. Data on
LUC expression in hundreds of independent tagged lines will be presented. This T-DNA trapping
technology is currently being applied to isolate inducible genes and promoters.
