A study of disease symptom development in cultured cells of Nicotiana tabacum L. var. Samsun NN resulting from the direct cellular injection of tobacco mosaic virus

Callus tissue of Nicotiana tabacum var. Samsun NN, a local lesion host of tobacco mosaic virus (TMV), was grown in liquid shake cultures. Three cell chains consisting of 2 to 4 cells each were transferred from the shake cultures to each of 3 different oil chamber microscope slides. In each slide culture a single cell of one chain was directly injected with an aqueous suspension of common TMV that had been passed through a Millipore bacterial filter. A single cell of another chain was injected with a portion of the same inoculum, which had been made noninfective by heating. A third cell chain served as an uninoculated control. Injections were made with a hollow glass needle connected by plastic tubing to a microinjection syringe. The injections were performed with the aid of a micromanipulator. Within 15 minutes after each injection a deposit of material formed at the periphery of the puncture surrounding the base of the needle tip. During the next 36 to 48 hours a thick layer of material was deposited over the entire needle tip, reestablishing the integrity of the cell wall. This permitted removal of the needle without loss of cell contents. ..