title: Assessment of genetic and epigenetic variation during long-term Taxus cell culture.
abstract: Gradual loss of secondary metabolite production is a common obstacle in the development of a large-scale plant cell production system. In this study, cell morphology, paclitaxel (Taxol®) biosynthetic ability, and genetic and epigenetic variations in the long-term culture of Taxus media cv Hicksii cells were assessed over a 5-year period to evaluate the mechanisms of the loss of secondary metabolites biosynthesis capacity in Taxus cell. The results revealed that morphological variations, gradual loss of paclitaxel yield and decreased transcriptional level of paclitaxel biosynthesis key genes occurred during long-term subculture. Genetic and epigenetic variations in these cultures were also studied at different times during culture using amplified fragment-length polymorphism (AFLP), methylation-sensitive amplified polymorphism (MSAP), and high-performance liquid chromatography (HPLC) analyses. A total of 32 primer combinations were used in AFLP amplification, and none of the AFLP loci were found to be polymorphic, thus no major genetic rearrangements were detected in any of the tested samples. However, results from both MSAP and HPLC indicated that there was a higher level of DNA methylation in the low-paclitaxel yielding cell line after long-term culture. Based on these results, we proposed that accumulation of paclitaxel in Taxus cell cultures might be regulated by DNA methylation. To our knowledge, this is the first report of increased methylation with the prolongation of culture time in Taxus cell culture. It provides substantial clues for exploring the gradual loss of the taxol biosynthesis capacity of Taxus cell lines during long-term subculture.
  
  
  CONCLUSIONS
  DNA methylation maybe involved in the regulation of paclitaxel biosynthesis in Taxus cell culture.
authors.affiliation: Department of Biotechnology, College of Life Science and Technology, Institute of Resource Biology and Biotechnology, Huazhong University of Science and Technology, 430074 Wuhan, China.
authors.forename: Chunhua
authors.initials: C
authors.lastname: Fu
authors.forename: Liqin
authors.initials: L
authors.lastname: Li
authors.forename: Wenjuan
authors.initials: W
authors.lastname: Wu
authors.forename: Maoteng
authors.initials: M
authors.lastname: Li
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Yu
authors.forename: Longjiang
authors.initials: L
authors.lastname: Yu
mesh_terms: D054458:Amplified Fragment Length Polymorphism Analysis; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D019175:DNA Methylation; D018744:DNA, Plant / Q000235:genetics; D044127:Epigenesis, Genetic; D014644:Genetic Variation; D017239:Paclitaxel / Q000096:biosynthesis; D059828:Plant Cells / Q000378:metabolism*; D020946:Taxus / Q000166:cytology / Q000235:genetics*; D013997:Time Factors
chemical_list: D018744:DNA, Plant; D017239:Paclitaxel