Inverted repeat sequences involved in high-frequency T-DNA truncation in “Princettia” (Euphorbia pulcherrima × Euphorbia cornastra)

Agrobacterium -mediated transformation is a primary method for gene transfer in plants and a crucial tool in plant biotechnology, generally leading to the integration of the entire T-DNA region into the plant genome. However, numerous plant species have documented undesirable T-DNA truncation during this integration. To address this, we investigated the sequence specificity of the truncation region using “Princettia,” an interspecific hybrid of poinsettia that exhibits this truncation at a high rate. Examination of 114 “Princettia” transformants revealed T-DNA truncation in 65 cases. These truncations occurred throughout the T-DNA, with certain regions showing higher frequencies. In a few lines, internal T-DNA sequences were missing, indicating multiple truncation events within a single T-DNA. Sequence analysis frequently identified inverted repeat (IR) sequences immediately flanking the T-DNA breakpoints. The highest truncation frequency was observed at a 113-bp IR sequence, suggesting that long IR sequences facilitate truncation, particularly those exceeding 100 bp. Additionally, partial insertions of inverted T-DNA regions were often found directly after the truncation sites, implying DNA polymerase involvement at these locations. Based on these observations, we propose a novel model where the binding of these IR sequences triggers truncation and the addition of extra sequences in their vicinity.