Liposome-mediated delivery of tobacco mosaic virus RNA into petunia protoplast : Improved conditions for liposome-protoplast incubations

Abstract

Liposome-mediated delivery of TMV RNA into petunia protoplasts and resulting virus antigen production has been used as an assay for determining incubation conditions which favor increased uptake of vesicle contents by plant cells. Vesicle phospholipid composition, incubation buffer divalent metal ion concentration, the type and concentration of polyalcohol used to stimulate vesicle uptake and the RNA content of the liposome preparation were determined to be critical factors influencing the efficiency of delivery. Manipulation of these parameters resulted in a 50-fold improvement in virus antigen production over that obtained with conditions previously used for liposome-protoplast incubations (Proc Natl Acad Sci 79: 1859-1863, 1982). Virus antigen production could be detected following incubation of protoplasts with <0.5 ng of encapsulated TMV RNA, while at higher concentrations of added liposomes, >80% of the protoplasts could be infected. Comparisons with other techniques used to introduce nucleic acids into plant protoplasts indicated that liposome-mediated delivery was 10-to 1 000-fold more efficient than these other methods. The general use of liposomes to introduce RNA and DNA molecules into plant protoplasts is discussed.