Comparative Analysis of Insect Resistance in Transgenic Populus × euramericana cv. Neva Expressing Dual Bt Genes from Different Sources

This study systematically evaluated insect resistance in transgenic poplar lines carrying three distinct <i>Bacillus thuringiensis</i> (<i>Bt</i>) gene vector architectures: a single-gene pb vector (<i>Cry1Ac</i>), a reverse-oriented double-gene n19 vector (<i>Cry1Ac</i>-<i>Cry3A</i>), and a forward-oriented double-gene n5 vector (<i>Cry3A</i>-<i>Cry1Ac</i>). The transgenic lines were accordingly designated as pb8/pb9, n19a/n19b, and DB7/DB16, respectively. Molecular analyses confirmed stable <i>Bt</i> gene integration, with the expression of <i>Cry3A</i> being consistently higher than that of <i>Cry1Ac</i> expression. Bioassays showed that dual-gene lines conferred broader insect resistance to pests than that of single-gene lines against both lepidopteran (<i>Hyphantria cunea</i>) and coleopteran (<i>Plagiodera versicolora</i>, <i>Anoplophora glabripennis</i>) pests. In contrast, the single-gene line pb9 exhibited specialized, high efficacy against <i>H. cunea</i>, achieving 100% mortality. Transcriptomic analysis of <i>P. versicolora</i> larvae fed the double-gene high-resistance n19a line and low-resistance DB16 line revealed multi-level molecular responses to Bt stress, including up-regulation of toxin-activating proteases, altered receptor expression, and suppression of growth-related genes. These changes were associated with significant developmental delay (8.33-20.83% reduction in the molting index). Our findings characterize the insect resistance and molecular profiles of the six transgenic poplar lines, as follows: multi-gene lines (n19a/n19b and DB7/DB16) confer broad-spectrum pest resistance, whereas single-gene lines (pb8/pb9) exhibit targeted efficacy. These results support the utility of these lines for pest-specific poplar breeding programs.