Specific Levels of DNA Methylation in Various Tissues, Cell Lines, and Cell Types of Daucus carota

Abstract

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).

Biological characterization of young and aged embryogenic cultures of Pinus pinaster (Ait.)

“…Comparison of methylated cytosines in the EM cultures of different ages, in both genotypes, showed values between 17.8% to 19.1% (standard error, 0.37) and a lack of significant difference (Table 5). This is in contrast to a study on carrot where the methylation levels of three cell suspension lines ranged from 14.5% to 22.6% (Palmgren et al 1991). The latter study also demonstrated that different cell types (vacuolar, meristematic, others) purified from the cultures had different methylation levels (from 22.2%, SD 0.2 to 25.4% SD 0.7); however, no statistical analysis of the data was provided to support this conclusion.…”

Changes in DNA methylation during somatic embryogenesis in Cucurbita pepo L.

“…DNA methylation is associated with gene inactivation (Cedar and Razin 1990;Palmgren et al 1991) and plays a role in gene activity and cell differentiation (Munksgaard et al 1995). In somatic embryogenesis of carrot the removal of auxin provokes a decrease in methylation followed by embryo development; methylation increases again in later embryo stages (Lo Schiavo et al 1989;Munksgaard et al 1995).…”

DNA methylation as a key process in regulation of organogenic totipotency and plant neoplastic progression?

“…In in vitro plant cultures, many differences in the methylation status have been observed between different cell types (Palmgren et al, 1991). Moreover, investigations with isoschizmneric restriction endonueleases differentially sensitive to methylation of recognition sites have demonstrated frequent methylation changes after tissue culture of somatic cells (Brown, 1989;Phillips, 1993: Phillips et al, 1994).…”