LIPOSOME-MEDIATED INTRODUCTION OF DNA INTO PLANT PROTOPLASTS AND CALLI

Liposome-mediated plant cell transformation offers several unique features, the most important of which is the protection of the nucleic acids against nucleases present in the plant cell media. Conditions for efficient fusion of liposomes and plant cell membranes, gene delivery and gene expression, were investigated using plasmids containing the chloramphenicol acetyl transferase (CAT) gene, and a cloned dimer of the tomato yellow leaf curl virus (TYLCV) genome, a geminivirus. Negatively charged large unilamellar vesicles were used to encapsulate the DNA and to introduce it into tobacco or tomato protoplasts and calli. CAT gene activity was monitored in tobacco protoplasts for at least 7 days after transformation. Plasmid DNA sequences were detectable for at least 9 days, apparently unmodified and unintegrated in the plant cell genome. TYLCV DNA was detectable in tomato protoplasts several days after inoculation, in its original dimeric form. In addition, molecules with sizes corresponding to the replicative form of the viral genome were found, suggesting that a unit-length viral genome was excised from the dimer and possibly began replicating in the tomato cells. The cloned TYLCV dimer, and the CAT gene, encapsulated in liposomes, were also used to inoculate wounded tomato and tobacco calli respectively. CAT activity was found in tobacco callus 14 days after inoculation, and cloned TYLCV dimer was detectable in tomato callus for 9 days after inoculation, apparently unmodified. However, only low molecular weight degradation products were found 14 days after inoculation. These results indicate that liposome-mediated gene transfer can be used to study transient gene expression and virus replication in plant cells.