ackground Development ornamental varieties with enhanced floral traits and distinctive characteristics is a central goal in floriculture. Anthocyanins, the principal flavonoid pigments in floral tissues, are synthesized via the well- characterized flavonoid biosynthesis pathway. Chalcone isomerase (CHI) catalyzes the first committed reaction in the flavonoid biosynthetic pathway. Method In this study, we investigated the effects of CHI suppression via RNA interference (RNAi) in three Petunia hybrida cultivars exhibiting distinct petal colors. Leaf discs from three Petunia hybrida lines exhibiting distinct petal colors (blue, pink, and purple) were inoculated with Agrobacterium tumefaciens harboring the recombinant plasmid pBI121 containing the RNAi construct targeting Phchi. Transgenic plants were regenerated on MS medium via microshoots emerging from wounded explant regions and verified by PCR using two primer pairs specific to the CHI silencing vector. Gene expression analysis was conducted for chi and associated pigment biosynthesis genes, including flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), flavonoid 3′,5′-hydroxylase (F3′5′H), and dihydroflavonol 4-reductase (DFR), comparing transgenic and wild-type plants. Chalcone and naringenin contents were quantified using a NanoDrop ELISA plate reader. Data analysis was performed using SPSS version 16, and mean comparisons were evaluated via T-test at a significance level of α = 0.05. Results Transformation efficiencies for CHI-silenced Petunia lines were 57.89%, 72.0% and 84.0% for the pink, blue, and purple phenotypes, respectively. Phenotypic evaluation revealed not only altered pigment distributions but also novel floral morphologies, such as tetramerous corollas and twisted tubular petal margins, in the CHI-suppressed lines. Quantitative metabolic profiling demonstrated a significant reduction in naringenin levels across all transgenic lines, whereas, chalcone accumulation was significantly elevated in a subset of lines.

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