http://www.dspace.espol.edu.ec/handle/123456789/1956 Tue, 07 Apr 2026 23:36:59 GMT 2026-04-07T23:36:59Z http://www.dspace.espol.edu.ec/handle/123456789/4827 Title: Promoter tagging in banana Authors: Remy, Serge; Coemans, Bert; Buysse, Sofie; Möller-Nielsen, Nina; Santos Ordoñez, Efren German; De Weerdt, Gerrit; Swennen, Rony; Sagi, Laszlo Abstract: 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. Sun, 01 Sep 2002 00:00:00 GMT http://www.dspace.espol.edu.ec/handle/123456789/4827 2002-09-01T00:00:00Z http://www.dspace.espol.edu.ec/handle/123456789/4825 Title: Isolation of plantain promoters using the firefly luciferase reporter gene Authors: Santos Ordoñez, Efren German; Remy, Serge; Coemans, Bert; Thiry, Else; Windelinckx, Saskia; Swennen, Rony; Sagi, Laszlo Abstract: For the isolation of promoters in plantain an Agrobacterium-mediated tagging approach was chosen. A promoterless luciferase reporter gene next to the left border of the T-DNA (pluc19) was introduced into embryogenic cells of the plantain ‘Three Hand Planty’. Five months after transformation, individual cell colonies in 24-well plates were screened in vitro for baseline luciferase activity. Positive lines showing activated luciferase expression were further screened at plantlet stage. The frequency of cultures with luciferase activity comparable to or higher than the CaMV35S promoter control was 0.15%. Cell lines were also screened at 8°C four months after transformation with a new T-DNA tagging construct containing an improved luciferase reporter gene next to the right border (pETKUL2). Screening of approximately 15,887 cell colonies revealed 155 (0.98%) with luciferase activation. Twenty three independent cell colonies (0.14%) showed responsive luciferase expression at 8°C. The isolation of flanking plant DNA sequences was accomplished by TAIL PCR; an average of four flanking sequences per line was obtained. Sequences were analyzed for conserved regions and putative transcription factor binding sites. Six out of 11 flanking T-DNA sequences of three promoter tagged lines showed vector backbone integration or T-DNA rearrangements. Five putative promoter sequences were isolated and analyzed, one of which turned out to be a putative GDP-mannose pyrophosphorylase gene. Thu, 01 Jul 2004 00:00:00 GMT http://www.dspace.espol.edu.ec/handle/123456789/4825 2004-07-01T00:00:00Z http://www.dspace.espol.edu.ec/handle/123456789/4824 Title: Characterization of promoter tagged lines Authors: Remy, Serge; Thiry, Else; Windelinckx, Saskia; Rymen, Bart; Santos Ordoñez, Efren German; Coemans, Bert; Swennen, Rony; Sagi, Laszlo 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. Thu, 01 Jul 2004 00:00:00 GMT http://www.dspace.espol.edu.ec/handle/123456789/4824 2004-07-01T00:00:00Z http://www.dspace.espol.edu.ec/handle/123456789/4826 Title: Promoter tagging in banana Authors: Remy, Serge; Coemans, Bert; Santos Ordoñez, Efren German; Buysse, Sofie; Möller-Nielsen, Nina; Cannaerts, Bart; Thiry, Else; Swennen, Rony; Sagi, Laszlo Abstract: For the identification and isolation of promoters and genes with specific expression patterns a T-DNA trapping technology has been assembled for banana, which allows in planta characterization of candidate promoters without a priori isolation of the corresponding genes. A promoterless luciferase reporter gene linked to the left T-DNA border has been introduced to embryogenic cell suspensions of several banana cultivars. Screening for luciferase activation has been performed without induction as well as via various induction treatments during early (two months) and later (four months) steps of in vitro regeneration. Early screening of approximately 35,000 transgenic colonies revealed 26 candidates (0.07%) with constitutive expression. In contrast, late screening of 2,788 proliferating cultures resulted in 12 cultures (0.43%) with constitutive expression comparable to the CaMV35S promoter. Further screening of transgenic colonies, proliferating cultures and in vitro plants has been performed under osmotic and aluminum stress conditions as well as after treatment with paraquat. Several positive lines have been regenerated into plants and high expression levels were confirmed in leaves and/or meristems. All candidates have been further propagated for plant regeneration and molecular analysis. Southern hybridization and sequence analysis demonstrated a range of 2-5 insertion sites in the genome with a frequent integration of the vector backbone or complex rearrangements. Therefore, RACE was used to specifically amplify the transcribed copies only and the analysis of 7 flanking regions isolated by TAIL-PCR will be presented. Improved tagging constructs have also been prepared which contain a codon-optimised luciferase gene with or without an intron fused next to the right T-DNA border. Sun, 01 Jun 2003 00:00:00 GMT http://www.dspace.espol.edu.ec/handle/123456789/4826 2003-06-01T00:00:00Z