[An integrated system for transient expression and stable transformation in Nicotiana benthamiana suspension cells]

Nicotiana benthamiana suspension cells, exhibiting advantages such as short growth cycles, facile genetic manipulation, ease of product purification, and suitability for large-scale cultivation, are emerging as a novel industrial biosynthesis platform for plant-derived natural products. Transient expression and stable transformation are core technologies for validating gene function and constructing plant cell factories. However, current methods have been developed primarily for whole-plant systems, systematic investigations in plant suspension cells are lacking, let alone a technological framework for integrating both approaches. Plant suspension cells, by virtue of cultivating characteristics, are suitable to develop integrated systems for transient expression and stable transformation. This study constructed an integrated system for transient expression and stable transformation (ISTS) by incorporating an efficient expression module, a transient expression enhancement module, and a stable integration screening module. ISTS enabled higher transient transformation efficiency and expression intensity in N. benthamiana suspension cells than classical pCAMBIA1300. Moreover, upon selection and cultivation of transiently expressing cells, stably transformed cell lines with efficient target gene integration and high expression levels can be obtained readily. In this study, tHMGR, tGGPPS, and tTS were cloned into ISTS vector individually and co-transformed into N. benthamiana suspension cells, which enabled the one-step multigene transient expression and stable integration. This approach successfully yielded taxadiene-producing tobacco cell lines. ISTS offers advantages such as simplified vector construction, fewer procedural steps, and shorter operational cycle. This work provides key technical support for establishing efficient, stable plant suspension cell factories and facilitating sustainable biosynthesis of complex natural products.