Tissue-specific activation tagging in Arabidopsis thaliana for identification and isolation of genes of agronomic importance

Activation tagging is used to recover and clone dominant gain-of-function alleles and usually employs a T-DNA vector containing four tandem copies of the CaMV35S enhancer sequence outward usually at the right border. This strategy, however, is not efficient as it could result in overexpression of multiple genes or genes far away from the insertion site. Therefore, we tested constitutive (CaMV35S) and tissue-specific (TFL1 and rbcS2B) promoters for activation tagging. From an initial screening of 400 T-1 plants, we identified several morphological variants which include seedling mutants (single cotyledon, slow seedling growth, long root, short root), leaf mutants (fused leaves, excess rosettes, altered shape, hyponastic leaves, bushy rosette, altered leaf polarity), inflorescence mutants (fasciated inflorescence, inflorescence fused with the petiole of rosette leaf) and flowering time and growth habit mutants (dwarf, tall, miniature, late flowering, sturdy stem). Comparison of different activation tagging populations for different categories of mutants revealed the pre-dominance of seedling mutants in the TFL1 promoter activation tagged population; leaf and inflorescence mutant frequencies were higher in rbcS2B promoter activation tagged population, while the flowering time and growth habit mutant frequency was higher in CaMV35S promoter activation tagged population. Flanking sequence analysis of three of the mutants showed that all the mutants carried an insertion in the intergenic region. Segregation analysis of seedlings for kanamycin resistance showed that on average each mutant carried about 1.3 copies of T-DNA.