Long-Term Potato Virus X (PVX)-Based Transient Expression of Recombinant GFP Protein in Nicotiana benthamiana Culture In Vitro

SIMPLE SUMMARY: Nowadays, the global recombinant protein market is valued at USD 125 billion. While the main producers of recombinant proteins are bacterial and mammalian cell cultures, plants have been intensively explored as an alternative platform for obtaining recombinant proteins (biopharming). Plants are safer systems, as they are free of human pathogens, oncogenic sequences, or bacterial endotoxins. Here, we proposed a new system for the production of recombinant proteins through transient expression technology in Nicotiana benthamiana plants grown in aseptic conditions (in vitro). Closed in vitro systems (cell tissue cultures) are well controlled; thus, they are preferable for recombinant protein production, as they can be more easily standardized for the pharmaceutical market purposes, and transient expression allows high levels of the recombinant proteins to be produced. We used an agrobacteria-delivered vector containing phytopathogenic virus (potato virus X) sequences to create plant tissue culture with prolongated transient expression of recombinant reporter green fluorescent protein (GFP). The mean of GFP was 18% of the total soluble cell proteins (TSP) (0.52 mg/g of fresh leaf weight (FW), and the best result reached 47% TSP (2 mg/g FW). The system can be a new technique for biopharming, combining the advantages of transient expression and cell tissue cultures. ABSTRACT: Plant molecular farming has a great potential to produce valuable proteins. Transient expression technology provides high yields of recombinant proteins in greenhouse-grown plants, but every plant must be artificially agroinfiltrated, and open greenhouse systems are less controlled. Here, we propose to propagate agrobacteria-free plants with high-efficient long-term self-replicated transient gene expression in a well-controlled closed in vitro system. Nicotiana benthamiana plant tissue culture in vitro, with transient expression of recombinant GFP, was obtained through shoot induction from leaf explants infected by a PVX-based vector. The transient expression occurs in new tissues and regenerants due to the natural systemic distribution of viral RNA carrying the target gene. Gene silencing was delayed in plants grown in vitro, and GFP was detected in plants for five to six months. Agrobacteria-free, GFP-expressing plants can be micropropagated in vitro (avoiding an agroinfiltration step), “rejuvenated” through regeneration (maintaining culture for years), or transferred in soil. The mean GFP in the regenerants was 18% of the total soluble proteins (TSP) (0.52 mg/g of fresh leaf weight (FW). The highest value reached 47% TSP (2 mg/g FW). This study proposes a new method for recombinant protein production combining the advantages of transient expression technology and closed cultural systems.