Hairy roots were used as an in vitro culture system for the propagation of wild-type and transgenic plant viruses. Tobacco mosaic virus (TMV) was added to the liquid culture medium at the same time as root inoculation. Hairy root growth was unaffected by viral infection. Maximum concentrations of TMV in Nicotiana benthamiana hairy roots were 1-2 orders of magnitude greater than in suspended N. benthamiana cells and reached levels of 1-2 mg g(-1) dry weight or 20-28% total soluble protein. Virus accumulated in the roots initially with a constant doubling time of about 1.0 day; subsequent reductions in viral growth rate were correlated with a significant decline in infectivity relative to the inoculum virus. The morphological integrity of the viral particles was maintained during propagation in hairy roots. The contribution to the overall viral titer of passive association of virus with the biomass, for example, by surface adsorption, was negligible compared with active viral replication. N. benthamiana hairy roots were also infected with a TMV-based viral vector developed to express green fluorescent protein (GFP). This vector was about 260-fold less infectious than wild-type TMV and accumulated much more slowly in the roots. Maximum levels of TMV-GFP in the biomass were about 65-fold lower than for TMV. This work demonstrates that hairy root cultures are a feasible means for in vitro propagation of wild-type and transgenic plant viruses under conditions that allow a high degree of environmental containment and control.

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Originally published in: Molecular Farming. Edited by Rainer Fischer and Stefan Schillberg. Copyright © 2004 Wiley‐VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3‐527‐30786‐9 The sections in this article are Foreign Protein Production Systems Production of Foreign Proteins Using Plant Tissue Culture Suspended Cell Cultures Hairy Root Cultures Shooty Teratoma Cultures Scale‐up Considerations for Different Forms of Plant Tissue Culture Strategies for Improving Foreign Protein Accumulation and Product Recovery in Plant Tissue Culture Expression Systems Modifications to Existing Expression Constructs Transient Expression Using Viral Vectors Secretion of Foreign Proteins Foreign Protein Stability Stability Inside the Cells Stability Outside the Cells Medium Additives Medium Properties Bioprocess Developments Product Recovery from the Medium Oxygen Transfer and Dissolved Oxygen Concentration Conclusions