Advances in T-DNA integration analysis: from classical methods to next-generation sequencing

Agrobacterium-mediated transformation leads to the integration of T-DNA into the plant genome. It is a critical event that determines the stability and expression of the integrated genes, and the regulatory acceptability of transgenic plants. The integration of T-DNA into the host plant genome occurs through various mechanisms during genetic transformation, with conventional detection methods primarily relying on PCR and Southern hybridization techniques. During T-DNA integration, the three-base extension of the right border sequence first identifies microhomology at a nick site within a chromosome, followed by the alignment of the left border microhomology. However, the left border extension may be absent in many transgenic plants due to incomplete T-DNA transfer, resulting in its partial integration into the plant genome. The integration process involves several steps, including the copying of T-DNA into nicks in the chromosomes and the search for microhomologies. This randomness can lead to chromosomal rearrangements, particularly when microhomologies occur between border sequences on different chromosomes, which limit the ability of conventional detection techniques to accurately pinpoint T-DNA loci. Next-Generation Sequencing (NGS) addresses these limitations through continuous advancements, particularly when integrated with CRISPR-Cas9 technology. This review aims to provide insights into the latest developments in technologies for detecting T-DNA integration in plants. By emphasizing these advancements, we seek to enhance the understanding of T-DNA integration mechanisms, which are crucial for improving the reliability and effectiveness of transgenic plant development.