Characterization and isolation of t-dna tagged banana promoters active during in vitro regeneration and low temperature stress

Abstract

A genome-wide T-DNA tagging strategy was pursued for the characterization and isolation of novel banana promoters. Banana embryogenic cell suspensions were transformed via Agrobacterium tumefaciens containing a promoterless, codon-optimized luciferase (luc+) gene next to the right T-DNA border. Approximately 89,000 transgenic banana cell colonies were screened for luminescence two to three months after transformation using a sophisticated camera system in a light-tight box. Screening occurred under controlled temperature conditions in which luminescence was monitored in real-time at 26°C followed by a gradual decrease to different low temperatures (LT) including 18°C, 16°C, 12°C and 8°C. The frequency of cell colonies showing luminescence at 26°C and the different LT treatments ranged from 0.17% to 2.69%. Transgenic cell colonies responsive to 8°C (i.e. showing an enhanced or repressed luciferase expression pattern) were regenerated to plantlets and luminescence was monitored at different in vitro developmental stages. With increasing differentiation the number of lines with LT up-regulated LUC expression decreased. Banana plants showing luminescence were analyzed for the T-DNA copy number by Southern hybridization. T-DNA inserts averaged 3.6 (range from 1 to 5) in 10 independent lines tested. DNA sequences flanking the right and left T-DNA borders were isolated via TAIL-PCR and inverse PCR. Continuity of sequence between the corresponding right and left border flanking sequences was revealed by linking PCR using flanking sequence specific primers. RT-PCR analysis was performed in lines with multiple inserts to identify and confirm the sequence that activated luciferase expression. Four candidate promoter sequences which were transcriptionally fused to the luc+ were cloned upstream of a uidAINT reporter gene and back-transformed to banana, which confirmed promoter activity for two sequences in in vitro cultures. Promoter activity in mature banana plants remains to be investigated. To summarize, a high-throughput T-DNA tagging system has enabled us to characterize and isolate novel banana promoters with potential for a number of downstream applications including banana improvement via genetic modification.