Effect of Different DNA Demethylating Agents on In vitro Cultures of Peach Rootstock GF 677

  • Miroslav BARÁNEK Mendel University in Brno, Faculty of Horticulture, Mendeleum – Institute of Genetics, Valtická 334, Lednice 69144 http://orcid.org/0000-0002-1583-3588
  • Miroslav OTMAR Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Flemingovo nám. 2, CZ-166 10, Prague 6
  • Marcela KREČMEROVÁ Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Flemingovo nám. 2, CZ-166 10, Prague 6
  • Aleš EICHMEIER Mendel University in Brno, Faculty of Horticulture, Mendeleum – Institute of Genetics, Valtická 334, Lednice 69144
  • Jana MOUDRÁ Mendel University in Brno, Faculty of Horticulture, Mendeleum – Institute of Genetics, Valtická 334, Lednice 69144
  • Zuzana MYNARZOVÁ Mendel University in Brno, Faculty of Horticulture, Mendeleum – Institute of Genetics, Valtická 334, Lednice 69144
Keywords: 5,6-dihydro-5-azacytosine; dihydroxypropyladenine; epigenetic changes; micropropagation; somaclonal variability


The appearance of somaclonal variability induced by in vitro cultivation is relatively frequent and, in some cases, provides a valuable source of new phenotypes suitable for crop improvement. Numerous studies have confirmed that these changes can be explained by alterations of DNA methylation. Interestingly, a group of chemical compounds termed ‘demethylating agents’ (DMT agents) enable artificial changes to be made in the DNA methylation state. Thus, these agents are theoretically able to induce new phenotypes or more favourable properties. The objective of the present study was to verify suitable conditions for the application of different DMT agents within in vitro protocols for micropropagation using the stone fruit rootstock GF 677 as an example. The impact of these agents on the properties of plant regenerants was evaluated, and their DNA methylation state was controlled by using an AFLP protocol based on a restriction endonuclease that differed in its sensitivity to methylated cytosines. Moreover, the effect of newly synthesised derivates was compared with that of conventional compounds with a well-documented DNA-demethylating impact. Based on the results, the suitable concentration for treatment by a DMT agent was established as approximately 50 µM. Promising results were generated using a combination of DMT agents with different mechanisms of action, such as azacytidine and dihydroxypropyladenine; under these conditions, probable synergy between methyltransferase interception by the cytosine analogue and interruption of methyl group donation by dihydroxypropyladenine significantly changed the DNA methylation state of treated plants. Regarding newly synthesised compounds, the 5,6-dihydro-5-azacytosine nucleoside showed the most promising results, which can likely be explained by its higher stability in the media used for in vitro cultivation.


Metrics Loading ...


Aghaye RNM, Yadollahi A (2012). Micropropagation of GF 677 rootstock. Journal of Agricultural Science 4(5):1916-9752.

Baránek M, Křižan B, Ondrušíková E, Pidra M (2010). DNA-methylation changes in grapevine somaclones following in vitro culture and thermotherapy. Plant Cell, Tissue and Organ Culture 101(1):11-22.

Baránek M, Čechová J, Raddová J, Holleinová V, Ondrušíková E, Pidra M (2015). Dynamics and reversibility of the DNA methylation landscape of grapevine plants (Vitis vinifera) stressed by in vitro cultivation and thermotherapy. PloS One 10:e0126638.

Baubec T, Pecinka A, Rozhon W, Mittelsten Scheid O (2009). Effective, homogeneous and transient interference with cytosine methylation in plant genomic DNA by zebularine. Plant Journal 57(3):542-554.

Bossdorf O, Arcuri D, Richards CL, Pigliucci M (2010). Experimental alteration of DNA methylation affects the phenotypic plasticity of ecologically relevant traits in Arabidopsis thaliana. Evolutionary Ecology 24(3):541-553.

Cheng JC, Matsen CB, Gonzales FA, Ye W, Greer S, Marquez F, … Selker EU (2003). Inhibition of DNA methylation and reactivation of silenced genes by zebularine. Journal of the National Cancer Institute 95(5):399-409.

Diesch J, Zwick A, Garz AK, Palau A, Buschbeck M, Götze KS (2016). A clinical-molecular update on azanucleoside-based therapy for the treatment of hematologic cancers. Clinical Epigenetics 8(1):71.

Dimasi-Theriou K, Economou AS (1995). Ethylene enhances shoot formation in cultures of the peach rootstock GF-677 (Prunus persica x P. amygdalus). Plant Cell Reports 15(1-2):87-90.

Fučík V, Michaelis A, Rigomar R (1970). On the induction of segment extension and chromatid structural changes in Vicia faba chromosomes after treatment with 5-azacytidine and 5-azadeoxycytidine. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 9(6):599-606.

Fulneček J, Kovařík A (2014). How to interpret Methylation Sensitive Amplified Polymorphism (MSAP) profiles? BMC Genetics 15:2.

Fraga HP, Vieira LN, Caprestano CA, Steinmacher DA, Micke GA, Spudeit DA, ... Guerra MP (2012). 5-Azacytidine combined with 2, 4-D improves somatic embryogenesis of Acca sellowiana (O. Berg) Burret by means of changes in global DNA methylation levels. Plant Cell Reports 31(12):2165-2176.

González APR, Dumalasová V, Rosenthal J, Skuhrovec J, Latzel V (2017). The role of transgenerational effects in adaptation of clonal offspring of white clover (Trifolium repens) to drought and herbivory. Evolutionary Ecology 31(3):345-361.

Hampl V, Pavlícek A, Flegr J (2001). Construction and bootstrap analysis of DNA fingerprinting-based phylogenetic trees with a freeware program FreeTree: Application to trichomonad parasites. International Journal of Systematic and Evolutionary Microbiology 51(3):731-735.

Karp A (1995). Somaclonal variation as a tool for crop improvement. Euphytica 85(1-3):295-302.

Kiselev KV, Tyunin AP, Manyakhin AY, Zhuravlev YN (2011). Resveratrol content and expression patterns of stilbene synthase genes in Vitis amurensis cells treated with 5-azacytidine. Plant Cell, Tissue and Organ Culture 105(1):65-72.

Kiselev KV, Ogneva ZV, Dubrovina AS, Nityagovsky NN, Suprun AR (2019). Somatic mutations, DNA methylation, and expression of DNA repair genes in Arabidopsis thaliana treated with 5-azacytidine. Biologia Plantarum 63:398-404.

Kovařík A, Koukalová B, Holý A, Bezdĕk M (1994). Sequence-specifc hypomethylation of the tobacco genome induced with dihydroxypropyladenine, ethionine and 5-azacytidine. FEBS Letters 353(3):309-311.

Krečmerová M, Otmar M (2012). 5-azacytosine compounds in medicinal chemistry: current stage and future perspectives. Future Medicinal Chemistry 4(8):991-1005.

Latzel V, Rendina González AP, Rosenthal J (2016). Epigenetic memory as a basis for intelligent behavior in clonal plants. Frontiers in Plant Science 7:1354.

Liu CH, Finke A, Díaz M, Rozhon W, Poppenberger B,... Pecinka A (2015). Repair of DNA damage induced by the cytidine analog zebularine requires ATR and ATM in Arabidopsis. The Plant Cell 27(6):1788-1800.

Matoušová M, Votruba I, Otmar M, Tloušťová E, Günterová J, Mertlíková-Kaiserová H (2011). 2'-deoxy-5,6-dihydro-5-azacytidine-a less toxic alternative of 2 '-deoxy-5-azacytidine A comparative study of hypomethylating potential. Epigenetics 6(6):769-776.

Mehta YR, Angra DC (2000). Somaclonal variation for disease resistance in wheat and production of dihaploids through wheat 9 maize hybrids. Genetics and Molecular Biology 23(3):617-622.

Nei M, Li WH (1979). Mathematical model for studying genetic variation in terms of restriction endonucleases. Proceedings of the National Academy of Sciences USA. 76(10):5269-5273.

Notari RE, DeYoung JL (1975). Kinetics and mechanisms of degradation of the antileukemic agent 5-azacytidine in aqueous solutions. Journal of Pharmaceutical Sciences 64(7):1148-57.

Popescu CF, Falk A, Glimelius K (2002). Application of AFLPs to characterize somaclonal variation in anther-derived grapevines. Vitis 41(4):177-182.

Predieri S. (2001) Mutation induction and tissue culture in improving fruits. Plant Cell, Tissue and Organ Culture 64(2-3):185-210.

Quoirin M, Lepoivre P (1977). Improved media for in vitro culture of Prunus sp. Acta Horticulturae (ISHS) 78:437-442.

Reuveni Y, Rosenthal LJ (1979). Effect of 5-azacytidine on cytoplasmic ribosomal and messenger ribonucleic acids in BSC-1 cells. Antimicrobial agents and chemotherapy 15(2): 235-239.

Sáez-Laguna E, Guevara MA, Díaz LM, Sánchez-Gómez D, Collada C, … Cervera MT (2014). Epigenetic variability in the genetically uniform forest tree species Pinus pinea L. PloS One 9(8): e103145.

Schanche JS, Schanche T, Ueland PM, Holý A, Votruba I (1984). The effect of aliphatic adenine analogues on S-adenosylhomocysteine and S-adenosylhomocysteine hydrolase in intact rat hepatocytes. Molecular Pharmacology 26(3):553-558.

Smulders MJM, De Klerk GJ (2011). Epigenetics in plant tissue culture. Plant Growth Regulation 63(2):137-146.

Stresemann C, Lyko F (2008). Modes of action of the DNA methyltransferase inhibitors azacytidine and decitabine. International Journal of Cancer 123(1):8-13.

Tsipouridis C, Thomidis T, Michailides Z (2005). Factors influencing the rooting of peach GF677 (peach ¥ almond hybrid) hardwood cuttings in a growth chamber. New Zealand Journal of Crop and Horticultural Science 33(2):93-98.

Unai E, IselenT, de Garcia E (2004). Comparison of characteristics of bananas (Musa sp.) from the somaclone CIEN BTA-03 and its parental clone Williams. Fruits 59(4):257-263.

Valledor L, Hasbún R, Meijón M, Rodríguez JL, Santamaría E, Viejo M, … Rodríguez R (2007). Involvement of DNA methylation in tree development and micropropagation. Plant Cell, Tissue and Organ Culture 91(2):75-86.

Vergeer P, Ouborg NJ (2012). Evidence for an epigenetic role in inbreeding depression. Biology Letters 8(5):798-801.

Yoo CB, Cheng JC, Jones PA (2004). Zebularine: a new drug for epigenetic therapy. Biochemical Society Transactions 32:910-912.

How to Cite
BARÁNEK, M., OTMAR, M., KREČMEROVÁ, M., EICHMEIER, A., MOUDRÁ, J., & MYNARZOVÁ, Z. (2019). Effect of Different DNA Demethylating Agents on In vitro Cultures of Peach Rootstock GF 677. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 47(3), 896-902. https://doi.org/10.15835/nbha47311373
Research Articles