Engineering potato inbred lines expressing Cry1Ac and Cry3A for dual resistance to potato tuber moth and Colorado potato beetle

Potato ( Solanum tuberosum ) is critical for global food security. However, potato production is severely constrained by two major pests, potato tuber moth ( Phthorimaea operculella , PTM) and Colorado potato beetle ( Leptinotarsa decemlineata , CPB), which can cause catastrophic yield losses of up to 90% under outbreak conditions. Developing durable, broad-spectrum resistance within genetically tractable breeding systems poses a major challenge to potato improvement. Here, we established an effective dual-resistance strategy centered on a diploid inbred platform by co-expressing codon-optimized Bacillus thuringiensis (Bt) genes cry1Ac and cry3A in the self-compatible diploid inbred line A6-56 ( Solanum tuberosum Group Stenotomum ), as well as various tetraploid cultivars, via Agrobacterium-mediated transformation. The engineered diploid line exhibited strong and stable resistance to both PTM and CPB, providing a simplified, genetically stable system for trait integration compared with using conventional tetraploid backgrounds. Crucially, reciprocal crosses between the transgenic diploid line and another inbred line, E4-63 ( Solanum tuberosum Group Phureja ), produced F 1 progeny that retained strong insect resistance and consistent transgene expression. These results demonstrate the stable Mendelian inheritance and functional conservation of Cry1Ac and Cry3A, underscoring the unique advantage of using diploid inbred lines for precise trait transmission and rapid deployment in breeding programs. Our findings provide both a powerful breeding resource and a scalable strategy for achieving durable, broad-spectrum insect resistance, which should advance the development of efficient, predictable, sustainable potato improvement systems.