DNA mutagenesis in 2- and 20-yr-old Panax ginseng cell cultures

  • Konstantin V. Kiselev
  • Alexandra S. Dubrovina
  • Olga A. Shumakova
Plant Tissue Culture


Previously, Panax ginseng var. Mimaki C.A. Meyer had been shown to accumulate genetic mutations during long-term propagation of a callus culture over a period of 20 yr. In this study, we analyzed the mutation types and frequency in a 2-yr-old P. ginseng callus culture and compared it with the 20-yr-old callus culture, and leaves of cultivated plants. We analyzed the sequence variability between the Actin genes, which are a family of housekeeping genes; phenylalanine ammonia-lyase (PAL) and dammarenediol synthase (DDS), which actively participate in the biosynthesis of ginsenosides; and the somatic embryogenesis receptor kinases (SERK), which control plant development. The frequency of point mutations in the Actin, PAL, DDS, and SERK genes in the 2-yr-old P. ginseng callus culture was markedly higher than in cultivated plants, but lower than in the 20-yr-old callus culture. Most of the mutations in the 2-yr-old P. ginseng calli were A↔G and T↔C transitions, as in the 20-yr-old calli and intact P. ginseng plants. The number of nonsynonymous mutations was higher in the 2- and 20-yr-old callus cultures than the number of nonsynonymous mutations in cultivated P. ginseng. Interestingly, the total number of N→G or N→C substitutions in the analyzed genes was 1.6 times higher than the total number of N→A or N→T substitutions. Using a methylation-sensitive DNA fragmentation assay, we showed the level of methylcytosine to be higher in the DNA of the 20-yr-old P. ginseng calli that than in the DNA of the 2-yr-old cultures.


Panax ginseng Cell cultures Mutagenesis Nucleotide substitutions 



This work was supported by grants from the Russian Foundation for Basic Research (10-04-00189-а) and from the Far East Division of the Russian Academy of Sciences (12-III-А-06-072).


  1. Bairu MW, Aremu AO, Van Staden J (2011) Somaclonal variation in plants: causes and detection methods. Plant Growth Regul 63:147–173CrossRefGoogle Scholar
  2. Ball DJ, Gross DS, Garrard WT (1983) 5-Methylcytosine is localized in nucleosomes that contain histone H1. P Natl Acad Sci USA 80:5490–5494CrossRefGoogle Scholar
  3. Barret P, Brinkman M, Beckert M (2006) A sequence related to rice Pong transposable element displays transcriptional activation by in vitro culture and reveals somaclonal variations in maize. Genome 49:1399–1407PubMedCrossRefGoogle Scholar
  4. Bregitzer P, Halbert SE, Lemaux PG (1998) Somaclonal variation in the progeny of transgenic barley. Theor Appl Genet 96:421–425CrossRefGoogle Scholar
  5. Bulgakov VP, Khodakovskaya MV, Labetskaya NV, Chernoded GK, Zhuravlev YN (1998) The impact of plant rolС oncogene on ginsenoside production by ginseng hairy root cultures. Phytochemistry 49:1929–1934CrossRefGoogle Scholar
  6. Carver BF, Johnson BB (1989) Partitioning of variation derived from tissue culture of winter wheat. Theor Appl Genet 78:405–410CrossRefGoogle Scholar
  7. Clegg MT, Cummings MP, Durbin ML (1997) The evolution of plant nuclear genes. P Natl Acad Sci USA 94:7791–7798CrossRefGoogle Scholar
  8. Dahleen LS, Stuthman DD, Rines HW (1991) Agronomic trait variation in oat lines derived from tissue culture. Crop Sci 31:90–94CrossRefGoogle Scholar
  9. Diéguez MJ, Vaucheret H, Paszkowski J, Mittelsten Scheid O (1998) Cytosine methylation at CG and CNG sites is not a prerequisite for the initiation of transcriptional gene silencing in plants, but it is required for its maintenance. Mol Gen Genet 259:207–215PubMedCrossRefGoogle Scholar
  10. Dollé ME, Snyder WK, Gossen JA, Lohman PH, Vijg J (2000) Distinct spectra of somatic mutations accumulated with age in mouse heart and small intestine. P Natl Acad Sci USA 97:8403–8408CrossRefGoogle Scholar
  11. Duncan RR (1997) Tissue culture-induced variation and crop improvement. Adv Agron 58:201–240CrossRefGoogle Scholar
  12. Garcia AM, Calder RB, Dollé ME, Lundell M, Kapahi P, Vijg J (2010) Age- and temperature-dependent somatic mutation accumulation in Drosophila melanogaster. PLoS Genet 6:e1000950PubMedCrossRefGoogle Scholar
  13. Hirochika H (1993) Activation of tobacco transposons during tissue culture. EMBO J 12:2521–2528PubMedGoogle Scholar
  14. Hirochika H, Sugimoto K, Otsuki Y, Tsugawa H, Kanda M (1996) Retrotransposons of rice involved in mutations induced by tissue culture. P Natl Acad Sci USA 93:7783–7788CrossRefGoogle Scholar
  15. Hsu TW, Tsai WC, Wang DP, Lin S, Hsiao YY, Chen WH, Chen HH (2008) Differential gene expression analysis by cDNA-AFLP between flower buds of Phalaenopsis Hsiang Fei cv. H. F. and its somaclonal variant. Plant Sci 175:415–422CrossRefGoogle Scholar
  16. Kaeppler SM, Kaeppler HF, Rhee Y (2000) Epigenetic aspects of somaclonal variation in plants. Plant Mol Biol 43:179–188PubMedCrossRefGoogle Scholar
  17. Khrolenko YA, Burundukova OL, Lauve LS, Muzarok TI, Makhan’kov VV, Zhuravlev YN (2012) Characterization of the variability of nucleoli in the cells of Panax ginseng Meyer in vivo and in vitro. J Ginseng Res 36:322–326CrossRefGoogle Scholar
  18. Kiselev KV, Tchernoded GK (2009) Somatic embryogenesis in the Panax ginseng cell culture induced by the rolC oncogene is associated with increased expression of WUS and SERK genes. Russ J Genet 45:445–452CrossRefGoogle Scholar
  19. Kiselev KV, Gorpenchenko TY, Tchernoded GK, Dubrovina AS, Grishchenko OV, Bulgakov VP, Zhuravlev YN (2008) Calcium-dependent mechanism of somatic embryogenesis in Panax ginseng cell cultures expressing the rolC oncogene. Mol Biol 42:243–252CrossRefGoogle Scholar
  20. Kiselev KV, Turlenko AV, Tchernoded GK, Zhuravlev YN (2009a) Nucleotide substitutions in rolC and nptII gene sequences during long-term cultivation of Panax ginseng cell cultures. Plant Cell Rep 28:1273–1278PubMedCrossRefGoogle Scholar
  21. Kiselev KV, Dubrovina AS, Bulgakov VP (2009b) Phenylalanine ammonia–lyase and stilbene synthase gene expression in rolB transgenic cell cultures of Vitis amurensis. Appl Microbiol Biotechnol 82:647–655PubMedCrossRefGoogle Scholar
  22. Kiselev KV, Turlenko AV, Zhuravlev YN (2010) Structure and expression profiling of a novel calcium-dependent protein kinase gene PgCDPK1a in roots, leaves, and cell cultures of Panax ginseng. Plant Cell Tiss Organ Cult 103:197–204CrossRefGoogle Scholar
  23. Kiselev KV, Shumakova OA, Tchernoded GK (2011) Mutation of Panax ginseng genes during long-term cultivation of ginseng cell cultures. J Plant Physiol 168:1280–1285PubMedCrossRefGoogle Scholar
  24. Lauve LS, Burundukova OL, Muzarok TI, Zhuravlev YN (2008) Chromosome numbers of Panax ginseng (Araliaceae). Bot J 93:158–161Google Scholar
  25. Miguel C, Marum L (2011) An epigenetic view of plant cells cultured in vitro: somaclonal variation and beyond. J Exp Bot 62:3713–3725PubMedCrossRefGoogle Scholar
  26. Noro Y, Takano-Shimizu T, Syono K, Kishima Y, Sano Y (2007) Genetic variations in rice in vitro cultures at the EPSPs–RPS20 region. Theor Appl Genet 114:705–711PubMedCrossRefGoogle Scholar
  27. Okamoto H, Hirochika H (2000) Efficient insertion mutagenesis of Arabidopsis by tissue culture-induced activation of the tobacco retrotransposon Tto1. Plant J 23:291–304PubMedCrossRefGoogle Scholar
  28. Pérez G, Mbogholi A, Sagarra F, Aragón C, González J, Isidrón M, Lorenzo JC (2011) Morphological and physiological characterization of two new pineapple somaclones derived from in vitro culture. In Vitro Cell Dev Biol Plant 47:428–433CrossRefGoogle Scholar
  29. Phillips RL, Kaeppler SM, Olhoft P (1994) Genetic instability of plant tissue cultures: breakdown of normal controls. P Natl Acad Sci USA 91:5222–5226CrossRefGoogle Scholar
  30. Rani V, Raina SN (2000) Genetic fidelity of organized meristem-derived micropropagated plants: a critical reappraisal. In Vitro Cell Dev Biol Plant 36:319–330CrossRefGoogle Scholar
  31. Rotskaya UN, Rogozin IB, Vasyunina EA, Malyarchuk BA, Nevinsky GA, Sinitsyna OI (2010) High frequency of somatic mutations in rat liver mitochondrial DNA. Mutat Res Fundam Mol Mech Mutagen 685:97–102CrossRefGoogle Scholar
  32. Sato M, Hosokawa M, Doi M (2011) Somaclonal variation is induced de novo via the tissue culture process: a study quantifying mutated cells in Saintpaulia. PLoS One 6:e23541PubMedCrossRefGoogle Scholar
  33. Shumakova OA, Manyakhin AY, Kiselev KV (2011) Resveratrol content and expression of phenylalanine ammonia-lyase and stilbene synthase genes in cell cultures of Vitis amurensis treated with coumaric acid. Appl Biochem Biotechnol 165:1427–1436PubMedCrossRefGoogle Scholar
  34. Tyunin AP, Kiselev KV, Zhuravlev YN (2012) Effects of 5-azacytidine induced DNA demethylation on methyltransferase gene expression and resveratrol production in cell cultures of Vitis amurensis. Plant Cell Tiss Organ Cult. doi: 10.1007/s11240-012-0125-x
  35. Wang Q, Wang M, Zhang X, Hao B, Kaushik SK, Pan Y (2011) WRKY gene family evolution in Arabidopsis thaliana. Genetica 139:973–983PubMedCrossRefGoogle Scholar
  36. Wang YP, Wang XY, Paterson AH (2012) Genome and gene duplications and gene expression divergence: a view from plants. Ann NY Acad Sci 1256:1–14PubMedCrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2012

Authors and Affiliations

  • Konstantin V. Kiselev
    • 1
  • Alexandra S. Dubrovina
    • 1
  • Olga A. Shumakova
    • 1
    • 2
  1. 1.Laboratory of Biotechnology, Institute of Biology and Soil ScienceFar East Branch of Russian Academy of SciencesVladivostokRussia
  2. 2.Department of Biochemistry and BiotechnologyFar Eastern Federal UniversityVladivostokRussia

Personalised recommendations