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Abstract
The propagation of wild-type virus and a transgenic viral vector was examined in cultured plant cells to identify factors affecting viral infection of and accumulation in cultured cells and to determine if viral vectors could be used to facilitate the expression of heterologous proteins in vitro.
Tobacco mosaic virus (TMV) accumulation was examined in Nicotiana tabacum and N. benthamiana suspension and hairy root cultures. TMV accumulation was superior in N. benthamiana hairy roots. Hairy roots were infected by adding TMV to the liquid culture medium at the same time as root inoculation. Hairy root growth was unaffected by virus infection. The distribution of virus within root mats from shake-flask grown cultures was non-uniform and the concentration of virus accumulated in replicate cultures was variable.
When N. benthamiana hairy roots were infected using 1.5 μg mL-1 TMV, the average maximum concentration of virus accumulated in the biomass was 1.6 ± 0.25 mg g-1 dry weight, or 15-fold lower than in TMV infected N. tabacum leaves. Virus coat protein accumulated to a level of (26 ± 10)% total soluble protein in the hairy roots. Inoculum virus concentration and the medium in which infection was performed affected the virus yield and the percentage of inoculated cultures that accumulated virus. When cultures were inoculated using 9.0 μg mL-1 TMV, virus accumulated in the biomass to 4.2 ± 0.60 mg g-1 dry weight. Proportional scale-up of hairy root infection in shake flasks did not result in constant virus concentrations in the scaled cultures. TMV accumulation in bioreactor-infected and -grown hairy roots was poor.
N. benthamiana hairy roots were infected with a TMV-based vector (30B-GFPC3) that encoded Cycle 3 green fluorescent protein (GFP). TMV-GFPC3 was (260 ± 140)-fold less infectious than TMV as measured by local lesion assays. Propagation of TMV-GFPC3 could not be confirmed using mass balance. GFP was not detected in the infected hairy roots or the culture medium.
Hairy roots represent a potentially viable culture-based system for the in vitro production of virus and virus products when field-grown agricultural systems do not adequately address issues of containment or product safety.