Abstract

The nature of T-DNA/Ds insertion decides the utility of launch pad lines for iAc/Ds based insertional mutagenesis. Direct or inverted T-DNA/Ds repeats and insertions with vector backbone lead to poor recovery of flanking sequences; whereas T-DNA/Ds insertion leading to gene trap would limit the use of such launch pad lines. A new Ds tagging/trapping vector, pNU435 containing two copies of intron-interrupted barnase was used in tomato to counter select such T-DAN/Ds insertions. T₁ and T₂ plants generated in this study were devoid of direct repeats, vector backbone and gene traps as evidenced by the lack of barnase expression. Further evidence based on the LB flanking sequence recovered through TAIL-PCR did not show any vector backbone or direct repeats. But two out of six plants showed inverted repeats. Genome search with LB flanking sequence indicated that the insertions were not in genic region, and hence not led to gene traps. pNU435 with features for counter selecting undesirable T-DNA/Ds insertions can be employed for high throughput functional genomics.

Key words: pNU435 vector, T-DNA/Ds insertion, barnase, tomato, flanking sequence, functional genomics.