The level of DNA methylation in Daucus carota was found to be tissue specific, but no simple correlation between developmental stage or age of tissue and the level of DNA methylation was found. Among three different suspension culture lines from the same variety grown under identical conditions, large differences in the level of DNA methylation were observed. The highest and lowest levels were found in two embryogenic cell lines originating from the same clone. Suspension cells from one of the embryogenic cell lines were fractionated into three morphologically defined cell types using Percoll gradient density centrifugation, and the uniformity of these fractions was evaluated by image analysis. The three cell types showed different levels of DNA methylation. The lowest level was found in the fraction containing the precursor cells of somatic embryos.(Uppsala, Sweden). The suspension culture cell lines N04, NR, and NR2 were established from seedlings of the "Nobo" variety (Danefeldt, Odense, Denmark). They were initiated and kept as previously described (14). Somatic embryos were obtained by washing cells three times in Gamborg B5 medium (7) without hormones and diluting the cells to 10% settled cell volume. They were then plated on the same medium with 0.7% (w/v) agarose (type I, Sigma) and kept in the light for 7 d or until globular somatic embryos had emerged.Isolation of DNA and Determination of the Level of DNA Methylation DNA was isolated and further purified and the level of DNA methylation was determined as previously described (14). Briefly, DNA was digested by nuclease P1 and bacterial alkaline phosphatase and the resulting deoxyribonucleosides were separated and quantified by HPLC. mdC2 is present in DNA ofall higher organisms investigated (5, 15). DNA methylation is involved in regulation of gene activity and differentiation, and normally methylation of a gene inactivates its transcription (4, 6). In plants, it has been shown that in tissues, where a gene is expressed, it is often hypomethylated in comparison to tissues, where it is not expressed (1, 2, 16).

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We have studied the effect of the demethylating agent azacytidine (azaC) on expression of a beta-glucuronidase (GUS) gene transferred to tobacco leaf disks by Agrobacterium-mediated transformation. In a system where no selection was performed, where shoot formation was partially repressed, and where Agrobacterium does not express the GUS gene, we were able to follow the early events of transient and stable expression. Two days after inoculation, 8% of the cells expressed GUS but this proportion rapidly decreased to near zero in the following week. Treatment of leaf disks with azaC just after transformation retarded this inactivation to some extent, while treatment of Agrobacterium prior to transformation increased the frequency of transient expression. Three weeks after inoculation the number of GUS-expressing cells increased 4- to 6-fold in the leaf disks treated with azaC and in the leaf disks transformed with azaC-treated bacteria, while the control remained low. These data suggest that DNA methylation is involved in transgene inactivation and that a large number of silent but potentially active transgenes become integrated.

In one of 30 transgenic tobacco (Nicotiana tabacum) plants, the expression of an introduced ,B-glucuronidase (GUS) gene driven by the cauliflower mosaic virus 35S promoter, was found to be repressed as the plant matured, whereas the endogenous GUS activity was unaffected. Plants grown from seeds or regenerated from leaf discs derived from this plant showed a similar temporal pattern of expression. Suspension-cultured cells established from nonexpressing leaves did not express the introduced gene. In these cells, the silent gene could be reactivated by treatment for 5 or 10 days with 5-azacytidine. Overall, demethylation of the genome preceded recovery of the enzyme activity. The increase in the fraction of reactivated cells progressed in two phases. Up to 8 weeks after starting the 5-azacytidine treatment, approximately 2 to 4% of the cells were expressing GUS, followed by a dramatic increase of GUS-expressing cells. Thirteen weeks after starting the 5-azacytidine treatment, the fraction of GUS-expressing cells amounted to 80%. At this time, the original overall level of DNA methylation was reestablished. The degree of DNA demethylation, as well as the magnitude of reactivation, was dependent on the duration of the 5-azacytidine treatment. These results demonstrate that DNA methylation appears to be involved in the regulation of the introduced GUS gene and that this development-dependent pattern of expression can be inherited.Among different transformants, a variation in the expression of introduced genes has often been detected (2,25). Such intertransformant variability has been explained by "positional effects" or a variety of epigenetic and genetic causes (11,18), such as DNA methylation.Methylation of cytosine residues in DNA is involved in the regulation of gene expression, and an inverse correlation between the transcriptional activity of certain genes and the level of methylation of these genes has been found (1, 5, 23). Tissue-specific patterns of methylation have been observed in gene sequences that encode tissue-specific functions (5,8,23 azaC is a cytidine analog, which, when incorporated into DNA, inhibits DNA methyltransferase activity (6,24). Apparently, azaC is able to activate genes in a selective manner rather than causing a global increase in gene expression, even though it is thought to act as a nonspecific DNA-demethylating agent (15). Here, we report the azaC-induced reactivation of a silent, introduced GUS gene. Because azaC is a specific inhibitor of DNA methylation, the demonstration of the azaC-induced reactivation of the gene indicates that DNA methylation should be involved in repression of the gene in cells grown in suspension and in the leaves from which these cells are derived. MATERIALS AND METHODS Plant Material and Growth ConditionsMature (flowering stage) primary transgenic plants, R1, of tobacco (Nicotiana tabacum L. cv Wisconsin 38) transformed with the Ti plasmid pMON9749 (10) were used. The transfer DNA of pMON9749 contains the NPTII gene (encoding NPTII) driven by ...