Assessment of age-related polyploidy in Quercus robur L. somatic embryos and regenerated plants using DNA flow cytometry

Flow cytometric analysis with 4,6-diamidino-2-phenylindole (DAPI) staining was used to screen for chromosomal changes in Quercus robur during in vitro culture. The initiated cell lines (1992 until 1999) were maintained via se condary embryogenesis on P24 medium with 0.9 muM 6-benzylaminopurine (BAP) in regular subculture intervals of 6 weeks. Regenerated plants established in the greenhouse and in vitro plantlets derived from encapsulated somatic embryos were screened. The embryogenic cell lines were characterized as ind ividual clones by isoenzyme analysis. Flow cytometric relative DNA content analysis of the first screening period revealed that somaclonal variation i n form of tetraploidy occurred in two out of 26 tested somatic embryo clone s (Alt and Jung). These two clones lost their ability to convert into plant lets. Intraspecific relative DNA content variation including technical vari ation was below 3%. In the second screening period, however, 3 out of 37 cl ones (Alt, E4.31H9 and P3.27H) contained tetraploid cells leading to the as sumption that the frequency of tetraploidy seems to be correlated with the duration of in vitro culture. No chromosomal differences were detected in r egenerated plants. However, tetraploidy occurred in 8% of the tested clones over a culture period of 7 years.