Tissue-specific Activation Tagging in Arabidopsis thaliana for Identification and Isolation of Genes of Agronomic Importance

Activation tagging is used to recover and clone domi-nant 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 T1 plants, we identi-fied several morphological variants which include seedling mutants (single cotyledon, slow seedling growth, long ischolar_main, short ischolar_main), leaf mutants (fused leaves, excess rosettes, altered shape, hyponastic leaves, bushy rosette, altered leaf polarity), inflores-cence 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 popu-lation; leaf and inflorescence mutant frequencies were higher in rbcS2B promoter activation tagged popula-tion, while the flowering time and growth habit mutant frequency was higher in CaMV35S promoter activation tagged population. Flanking sequence ana-lysis of three of the mutants showed that all the mu-tants carried an insertion in the intergenic region. Segregation analysis of seedlings for kanamycin resis-tance showed that on average each mutant carried about 1.3 copies of T-DNA.