| Popis: |
Callus cells of tobacco ( Nicotiana tabacum L. var. Havana 38) were inoculated by mechanical dispersion with 12 μg/ml of tobacco mosaic virus (TMV) and were washed and incubated in a modified Murashige and Skoog (MS) liquid medium. Infectivity assays showed loss in cell-associated titer 10 hr after inoculation, followed by a sharp rise to 48 hr. Virus titer was maximal after 144 to 192 hr and thereafter declined. Maximal titer of cell-associated virus was 650 times that at 0 time and 1000 times that after 10 hr of inoculation. The amount of virus released by cells into the medium was greatest at 240 hr and represented 0.15% of cell-associated virus. Yields of virus, purified by differential centrifugation and estimated spectrophotometrically, were higher in cells incubated on agar than in suspension. Virus yields from inoculated cells incubated 7 days on agar were equal to those obtained from leaves of tobacco plants grown in soil. Cultured cells inoculated with 12 and 83 μg of TMV/ml yielded 1.27 and 2.20 mg virus per gram fresh weight of callus, respectively, or 1.0 to 1.7 × 10 7 virus particles per cell. Titers attained in a green-pigmented cell line were twice those attained in the ordinary, nonpigmented callus. Suspension-grown cells, inoculated by cell dispersion and incubated on agar yielded a titer 367% greater than agar-grown cells inoculated without cell dispersion and incubated in liquid medium. It is likely that cell dispersion causes nonvital injury to cells allowing virus particles avenues of entry. Probable sites of entry are ruptured plasmodesmatal connections. |
