DNA methylation and tissue culture-induced variation in plants

  • S. M. Kaeppler
  • R. L. Phillips
Somatic Cell Genetics/Genetic Transformation


Plant cells growing in an artificial culture environment make numerous genetic mistakes. These alterations are manifested as increased frequencies of single-gene mutations, chromosome breakages, transposable element activations, quantitative trait variations, and modifications of normal DNA methylation patterns. Evidence is presented that indicates a high frequency of DNA hypomethylation as the result of the tissue culture process. Fifteen percent of the methylation changes appear to have been homozygous in the original regenerated plants. A hypothesis is advanced that relates DNA methylation to the variety of genetic alterations found among maize tissue culture regenerants and their progenies. The epigenetic nature of DNA methylation raises questions concerning the stability of tissue culture-induced changes in self-pollinations and crosses.

Key words

tissue culture corn DNA methylation somaclonal variation 


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  1. Antequera, F.; Bird, A. P. Unmethylated CpG islands associated with genes in higher plants. EMBO J. 7:2295–2299; 1989.Google Scholar
  2. Armstrong, C. L. Genetic and cytogenetic stability of maize tissue cultures: a comparative study of organogenic and embryogenic cultures. Minneapolis: Univ. of Minnesota; 1986. Thesis.Google Scholar
  3. Armstrong, C. L.; Phillips, R. L. Genetic and cytogenetic variation in plants regenerated from organogenic and friable, embryogenic tissue cultures of maize. Crop Sci. 28:363–369; 1988.CrossRefGoogle Scholar
  4. Baenziger, P. S.; Wesenberg, D. M.; Schaeffer, G. W., et al. Variation among anther culture derived doubled haploids of “Kitt” wheat. In: Sakamoto, S., ed. Proceedings of the Sixth International Wheat Genetics Symposium. Kyoto, Japan: Plant Germ-Plasm Inst. for Agric., Kyoto Univ.; 1983.Google Scholar
  5. Banks, J. A.; Masson, P.; Fedoroff, N. Molecular mechanisms in the developmental regulation of the maize suppressor-mutator transposable element. Genes Dev. 2:1364–1380; 1988.PubMedGoogle Scholar
  6. Bayliss, M. W. Chromosomal variation in plant tissues in culture. Int. Rev. Cytol. Suppl. 11A:113–144; 1980.Google Scholar
  7. Benzion, G. Genetic and cytogenetic analysis of maize tissue cultures: a cell line pedigree analysis. Minneapolis: Univ. of Minnesota; 1984. Thesis.Google Scholar
  8. Benzion, G.; Phillips, R. L.; Rines, H. W. Case histories of genetic variabilityin vitro: oats and maize. In: Vasil, I. K., ed. Cell culture and somatic cell genetics of plants, vol. 3. New York: Academic Press; 1986:435–448.Google Scholar
  9. Bianchi, A.; Salamini, F.; Parlavecchio, R. On the origin of controlling elements in maize. Genet. Agrar. 22:335–344; 1969.Google Scholar
  10. Boyes, J.; Bird, A. DNA methylation inhibits transcription indirectly via a methyl-CpG binding protein. Cell 64:1123–1134; 1991.PubMedCrossRefGoogle Scholar
  11. Brettell, R. I. S.; Dennis, E. S. Reactivation of a silent Ac following tissue culture is associated with heritable alterations in its methylation pattern. Mol. Gen. Genet. 229:365–372; 1991.PubMedCrossRefGoogle Scholar
  12. Brettell, R. I. S.; Dennis, E. S.; Scowcroft, W. R., et al. Molecular analysis of a somaclonal variant of alcohol dehydrogenase. Mol. Gen. Genet. 202:335–344; 1986.CrossRefGoogle Scholar
  13. Brown, P. T. H. DNA methylation in plants and its role in tissue culture. Genome 31:717–729; 1989.Google Scholar
  14. Brown, P. T. H.; Kyozuka, J.; Sukekiyo, Y., et al. Molecular changes in protoplast-derived rice plants. Mol. Gen. Genet. 223:324–328; 1990.PubMedCrossRefGoogle Scholar
  15. Brown, P. T. H.; Gobel, E.; Lorz, H. RFLP analysis ofZea mays callus cultures and their regenerated plants. Theor. Appl. Genet. 81:227–232; 1991.CrossRefGoogle Scholar
  16. Cedar, H. DNA methylation and gene activity. Cell 53:3–4; 1988.PubMedCrossRefGoogle Scholar
  17. Chandler, V. L.; Walbot, V. DNA modification of a maize transposable element correlates with loss of activity. Proc. Natl. Acad. Sci. USA 83:1767–1771; 1986.PubMedCrossRefGoogle Scholar
  18. Chomet, P. S.; Wessler, S.; Dellaporta, S. Inactivation of the maize transposable element activator (Ac) is associated with its DNA modification. EMBO J. 6:295–302; 1987.PubMedGoogle Scholar
  19. Culley, D. E. Evidence for activation of a cryptic transposable element Ac in maize endosperm cultures. In: VI Int. Congr. Plant Tiss. Cell Culture, Minneapolis, MN. 3–8 August, 1986. St. Paul, MN: Univ. of Minnesota. Abstracts p.220.Google Scholar
  20. Dahleen, L. S.; Stuthman, D. D.; Rines, H. W. Agronomic trait variation in oat lines derived from tissue culture. Crop Sci. 31:90–94; 1991.CrossRefGoogle Scholar
  21. D’Amato, F. Cytogenetics of differentiation in tissue and cell cultures. In: Reinert, J.; Bajaj, V. P. S., eds. Plant cell, tissue, and organ culture. New York: Springer-Verlag; 1977:343–357.Google Scholar
  22. D’Amato, F. Cytogenetics of plant cell and tissue cultures and their regenerates. CRC Crit. Rev. Plant Sci. 3:73–112; 1985.Google Scholar
  23. Dennis, E. S.; Brettell, R. I. S.; Peacock, W. J. A tissue culture inducedAdh1 null mutant of maize results from a single base change. Mol. Gen. Genet. 210:181–183; 1987.CrossRefGoogle Scholar
  24. Doerschug, E. B. Studies ofDotted, a regulatory element in maize. I. Induction ofDotted by chromatid breaks. II. Phase variation ofDotted. Theor. Appl. Genet. 43:182–189; 1973.CrossRefGoogle Scholar
  25. Earle, E. B.; Gracen, V. E. Somaclonal variation in progeny of plants from corn tissue culture. In: Henke, R.; Hughes, K.; Hollaender, A., eds. Propagation of higher plants through tissue culture. New York: Plenum Press; 1985.Google Scholar
  26. Edallo, S.; Zucchinali, C.; Perenzin, M., et al. Chromosomal variation and frequency of spontaneous mutation associated within vitro culture and plant regeneration of maize. Maydica 26:39–56; 1981.Google Scholar
  27. Ergle, D. R.; Katterman, F. R. H. Deoxyribonucleic acid of cotton. Plant Physiol. 36:811–815; 1961.PubMedGoogle Scholar
  28. Evola, S. V.; Burr, F. A.; Burr, B. The nature of tissue culture-induced mutations in maize. Eleventh Annual Aharon Katzir-Katchalsky Conference, abstract.Google Scholar
  29. Fedoroff, N.; Banks, J.; Masson, P. Molecular genetic analysis of the maize suppressor-mutator element’s epigenetic developmental regulatory mechanism. Genome 31:973–979; 1989.Google Scholar
  30. Fukui, K. Sequential occurrence of mutations in a growing rice callus. Theor. Appl. Genet. 65:225–230; 1983.CrossRefGoogle Scholar
  31. Groose, R. W.; Bingham, E. T. An unstable anthocyanin mutation recovered from tissue culture of alfalfa (Medicago sativa). 1. High frequency of reversion upon reculture. 2. Stable nonrevertants derived from reculture. Plant Cell Rep. 5:104–110; 1986.CrossRefGoogle Scholar
  32. Gruenbaum, Y.; Naveh-Many, T.; Cedar, H. Sequence specificity of methylation in higher plant DNA. Nature 292:860–862; 1981.PubMedCrossRefGoogle Scholar
  33. Hall, G.; Allen, G. C.; Loer, D. S., et al. Nuclear scaffolds and scaffold-attachment regions in higher plants. Proc. Natl. Acad. Sci. USA 88:9320–9324; 1991.PubMedCrossRefGoogle Scholar
  34. Holliday, R. The inheritance of epigenetic defects. Science 238:163–170; 1987.PubMedCrossRefGoogle Scholar
  35. Johnson, S. S.; Phillips, R. L.; Rines, H. W. Possible role of heterochromatin in chromosome breakage induced by tissue culture in oats (Avena sativa L.). Genome 29:439–446; 1987.Google Scholar
  36. Kaeppler, S. M. Molecular and genetic studies of tissue culture-induced variation in maize. St. Paul: Univ. Minnesota; 1992. Thesis.Google Scholar
  37. Klaas, M.; Amasino, R. DNA methylation is reduced in DNase1-sensitive regions of plant chromatin. Plant Physiol. 91:451–454; 1989.PubMedGoogle Scholar
  38. Larkin, P. J. Somaclonal variation: history, method, and meaning. Iowa State J. Res. 61:393–434; 1987.Google Scholar
  39. Larkin, P. J.; Ryan, S. A.; Brettell, R. I. S., et al. Heritable somaclonal variation in wheat. Theor. Appl. Genet. 67:443–455; 1984.CrossRefGoogle Scholar
  40. Larkin, P. J.; Scowcroft, W. R. Somaclonal variation—a novel source of variability from cell cultures for plant improvement. Theor. Appl. Genet. 60:197–214; 1981.CrossRefGoogle Scholar
  41. Larkin, P.; Scowcroft, W. R. Somaclonal variation and crop improvement. In: Kosuge, T.; Meredith, C. P.; Hollander, A., eds. Genetic engineering of plants: an agricultural perspective. New York: Plenum Press; 1983:289–314.Google Scholar
  42. Lee, M. L.; Geadelmann, J. L.; Phillips, R. L. Agronomic evaluation of inbred lines derived from tissue cultures of maize. Theor. Appl. Genet. 75:841–849; 1988.CrossRefGoogle Scholar
  43. Lee, M. L.; Phillips, R. L. Genomic rearrangements in maize induced by tissue culture. Genome 29:122–128; 1987a.Google Scholar
  44. Lee, M. L.; Phillips, R. L. Genetic variability in progeny of regenerated maize (Zea mays L.) plants. Genome 29:344–355; 1987b.Google Scholar
  45. Lee, M. L.; Phillips, R. L. The chromosomal basis of somaclonal variation. Ann. Rev. Plant Physiol. Plant Mol. Biol. 39:413–437; 1988.CrossRefGoogle Scholar
  46. LoSchiavo, F.; Pitto, L.; Giuliano, G., et al. DNA methylation of embryogenic carrot cell cultures and its variations as caused by mutation, differentiation, hormones, and hypomethylating drugs. Theor. Appl. Genet. 77:325–331; 1989.CrossRefGoogle Scholar
  47. McClintock, B. The origin and behavior of mutable loci in maize. Proc. Natl. Acad. Sci. USA 36:344–355; 1950.PubMedCrossRefGoogle Scholar
  48. McClintock, B. Mechanisms that rapidly reorganize the genome. Stadler Genet. Symp. 10:25–47; 1978.Google Scholar
  49. McClintock, B. The significance of responses of the genome to challenge. Science 226:792–801; 1984.PubMedCrossRefGoogle Scholar
  50. McCoy, T. J.; Phillips, R. L.; Rines, H. W. Cytogenetic analysis of plants regenerated from oat (Avena sativa) tissue cultures; high frequency of partial chromsome loss. Can. J. Genet. Cytol. 24:37–50; 1982.Google Scholar
  51. McCoy, T. J.; Phillips, R. L. Chromosome stability in maize (Zea mays) tissue cultures and sectoring in some regenerated plants. Can. J. Genet. Cytol. 24:559–565; 1982.Google Scholar
  52. Messenguer, R.; Ganal, M. W.; Steffens, J. C., et al. Characterization of the level, target sites and inheritance of cytosine methylation in tomato nuclear DNA. Plant Mol. Biol. 16:753–770; 1991.CrossRefGoogle Scholar
  53. Monk, M. Changes in DNA methylation during mouse embryonic development in relation to X-chromosome activity and imprinting. Philos. Trans. R. Soc. Lond. B Biol. Sci. 326:299–312; 1990.PubMedGoogle Scholar
  54. Müller, E.; Brown, P. T. H.; Hartke, S., et al. DNA variation in tissue culture-derived rice plants. Theor. Appl. Genet. 80:673–679; 1990.Google Scholar
  55. Neuffer, M. G. Stability of the suppressor element in two mutator systems of theA1 locus in maize. Genetics 53:541–549; 1966.PubMedGoogle Scholar
  56. Oono, K.In vitro methods applied to rice. In: Thorpe, T. A., ed. Plant tissue culture. New York: Academic Press; 1981:273–298.Google Scholar
  57. Oono, K. Putative homozygous mutants in regenerated plants of rice. Mol. Gen. Genet. 198:377–384; 1985.CrossRefGoogle Scholar
  58. Orton, T. J. Genetic variation in somatic tissues: Method or madness? Adv. Plant Pathol. 2:153–189; 1984.Google Scholar
  59. Pardue, M. L. Dynamic instability of chromosomes and genomes. Cell 66:427–431; 1991.PubMedCrossRefGoogle Scholar
  60. Pavlica, M.; Nagy, B.; Papes, D. 2,4-D causes chromosome and chromatin abnormalities in plant cells and mutation in cultured mammalian cells. Mutat. Res. 263:77–82; 1991.PubMedCrossRefGoogle Scholar
  61. Peschke, V. M.; Phillips, R. L. Activation of the maize transposable elementSuppressor-mutator (Spm) in tissue culture. Theor. Appl. Genet. 81:90–97; 1991.CrossRefGoogle Scholar
  62. Peschke, V. M.; Phillips, R. L.; Gengenbach, B. G. Discovery of transposable element activity among progeny of tissue culture-derived maize plants. Science 238:804–807; 1987.CrossRefPubMedGoogle Scholar
  63. Peschke, V. M.; Phillips, R. L.; Gengenbach, B. G. Genetic and molecular analysis of tissue culture-derivedAc elements. Theor. Appl. Genet. 82:121–129; 1991.CrossRefGoogle Scholar
  64. Peschke, V. M.; Phillips, R. L. Genetic implications of somaclonal variation in plants. Adv. Genet. 30:41–75; 1992.CrossRefGoogle Scholar
  65. Peterson, P. A. A mutable plae green locus in maize. Genetics 38:682–683; 1953.Google Scholar
  66. Phillips, R. L. Somaclonal and gametoclonal variation. Genome 31:1119–1120; 1989.Google Scholar
  67. Phillips, R. L.; Kaeppler, S. M.; Peschke, V. M. Do we understand somaclonal variation? In: Nijkamp, H. J. J.; VanDerPlas, L. H. W.; Van Aartrijk, J., eds. Progress in plant cellular and molecular biology. Dordrecht: Kluwer Academic Publishing; 1990:131–141.Google Scholar
  68. Pryor, A.; Faulker, K.; Rhoades, M. M., et al. Asynchronous replication of heterochromatin in maize. Proc. Natl. Acad. Sci. USA 77:6705–6709; 1980.PubMedCrossRefGoogle Scholar
  69. Quemeda, H.; Roth, E. J.; Lark, K. G. Changes in methylation of tissue cultured soybean cells detected by digestion with the restriction enzymesHpall andMspI. Plant Cell Rep. 6:63–66; 1987.CrossRefGoogle Scholar
  70. Rhoades, M. M.; Dempsey, E. On the mechanism of chromatin loss induced by the B chromosome of maize. Genetics 71:73–96; 1972.PubMedGoogle Scholar
  71. Rhoades, M. M.; Dempsey, E. Chromatin elimination induced by the B chromosome of maize. J. Hered. 64:12–18; 1973.Google Scholar
  72. Rhodes, C. A.; Phillips, R. L.; Green, C. E. Cytogenetic stability of aneuploid maize tissue cultures. Can. J. Genet. Cytol. 28:374–384; 1986.Google Scholar
  73. Rice, T. B. Tissue culture induced genetic variation in regenerated maize inbreds. In: Proc. 37th Annu. Corn and Sorghum Research Conference. Chicago, IL, December, 1982. Washington, DC: Amer. Seed Assoc.; 1982:148–162.Google Scholar
  74. Schaeffer, G. W. Recovery of heritable variability in anther-derived doubled-haploid rice. Crop Sci. 22:1160–1164; 1982.CrossRefGoogle Scholar
  75. Schwartz, D. Gene-controlled cytosine demethylation in the promoter region of theAc transposable element in maize. Proc. Natl. Acad. Sci. USA 86:2789–2793; 1989.PubMedCrossRefGoogle Scholar
  76. Schwartz, D.; Dennis, E. Transposase activity of theAc controlling element in maize is regulated by its degree of methylation. Mol. Gen. Genet. 205:476–482; 1986.CrossRefGoogle Scholar
  77. Silva, A. J.; White, R. Inheritance of allelic blueprints for methylation patterns. Cell 54:145–152; 1988.PubMedCrossRefGoogle Scholar
  78. Sun, Z. X.; Zheng, K. L. Somaclonal variation in rice. In: Bajaj, Y. P. S., ed. Biotechnology in agriculture and forestry, vol. 3. Berlin: Springer-Verlag; 1990:288–325.Google Scholar
  79. Sunderland, N. Nuclear cytology. In: Street, H. E., ed. Plant tissue and cell culture, 2nd ed. Oxford: Blackwell; 1973:177–205.Google Scholar
  80. Vergara, R.; Verde, F.; Pitto, L., et al. Reversible variations in the methylation pattern of carrot DNA during somatic embryogenesis. Plant Cell Rep. 8:697–700; 1990.CrossRefGoogle Scholar
  81. Vincent, A.; Heitz, D.; Petit, C., et al. Abnormal methylation pattern detected in fragile-X patients by pulsed field gel electrophoresis. Nature 349:624–626; 1991.PubMedCrossRefGoogle Scholar
  82. Walbot, V. Reactivation of theMutator transposable element system following gamma irradiation of seed. Mol. Gen. Genet. 212:259–264; 1988.CrossRefGoogle Scholar
  83. Woodman, J. C.; Kramer, D. A. The recovery of somaclonal variants from tissue cultures of B73, an elite inbred line of maize. In: VI Intl. Congr. Plant Tissue Cell Culture, Minneapolis, MN, 3–8 August, 1986. St. Paul, MN: Univ. of Minnesota. Abstracts, p. 215.Google Scholar
  84. Zehr, B. E.; Williams, M. E.; Duncan, D. R., et al. Somaclonal variation among the progeny of plants regenerated from callus cultures of seven inbred lines of maize. Can. J. Bot. 61:491–499; 1987.CrossRefGoogle Scholar

Copyright information

© Tissue Culture Association 1993

Authors and Affiliations

  • S. M. Kaeppler
    • 1
  • R. L. Phillips
    • 1
  1. 1.Department of Agronomy and Plant Genetics and Plant Molecular Genetics InstituteUniversity of MinnesotaSt. Paul
  2. 2.Department of AgronomyUniversity of NebraskaLincoln

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