A highly efficient method of inoculation of tobacco mesophyll protoplasts with ribonucleic acid of tobacco mosaic virus

Summary

A highly efficient method of inoculation of tobacco mesophyll protoplasts with the ribonucleic acid (RNA) of tobacco mosaic virus (TMV) is standardised. Instead of milligrams of TMV-RNA needed in earlier studies, only microgram amounts are sufficient, if freshly prepared protoplasts are suspended in an alkaline buffer (pH 9.0) having a relatively high salt concentration. No poly-L-ornithin is necessary for infection under the present experimental conditions. Some infection takes place with as little as 0.001 μg TMV-RNA/ml, but to achieve nearly 100% infection of protoplasts, RNA-concentration of 4–20 μg/ml is recommended. Under optimal conditions each infected protoplast produced 10⁶–10⁷ particles of TMV within 48 hours. The advantages of using free TMV-RNA instead of intact TMV as inoculum lie in the absence of residual infectivity and of viral antigens at zero time. The method may prove to be of a more universal applicability in attempts to transfer foreign genetic informations to plant cells in the form of free nucleic acids.