The percentage of cells successfully transformed by Agrobacteria is usually very low (not more than 10% and usually much less; Thomas et al., 1989). In this report we show that in carrot (Daucus carota L., ssp. sativus) cell suspensions transformation efficiency was strongly improved by using cell cycle synchronized cells. Fluorodesoxyuridine (FDU) was added for 24 h to inhibit thymidine synthesis. This blocked the cell cycle at the transition from G1- to S-phase. Then the block was released by applying thymidine. A high rate of transformation was obtained when Agrobacterium tumefaciens was added concurrently with thymidine. As examples of efficient and long term foreign gene expression in transgenic cells, the reporter enzyme β-glucuronidase (GUS) as a model as well as the major hepatitis B virus surface protein were used. Both genes were linked to the MAS promoter. In carrot cell suspensions containing the viral gene, the corresponding viral protein was produced. In roots of mature transgenic carrot plants generated through somatic embryogenesis and raised in soil as well as in callus cultures derived thereof, the viral protein was also produced.

In chronically infected patients, hepatitis B virus (HBV) particles reach numbers as large as >109 genome equivalents (GE)/ml of serum. However, expression of infectious HBV particles in cell culture only yields 105–106 GE/ml, which is insufficient for many studies. HBV transcription and possibly replication is dependent on hepatocyte-specific differentiation. Thus, we tested several cell culture parameters that have been reported to enhance the expression of hepatocyte-specific markers, such as growth on different extracellular matrices, different cell culture media, low concentrations of fetal calf serum (FCS) and the addition of dimethyl sulfoxide (DMSO) to the medium. Lower concentrations of FCS, growth on collagen and inclusion of DMSO in the medium only moderately enhanced HBV production in vitro when applied individually. However, combinations of these parameters optimised cell culture conditions and reproducibly increased the release of HBV particles about 100-fold to titres >108 GE/ml of culture medium.