Allelic state and effects of VRN genes on soft wheat in in vivo and in vitro systems

  • O. O. Aksentyeva Taras Shevchenko Kyiv National University
  • V. V. Shulik V.N. Karazin Kharkiv National University
Keywords: Triticum aestivum, NILs, VRN genetic system, rate of development, culture in vitro, frequency of callusogenesis, PCR analysis, allelic variants


The article investigates the effects of the main manifestations of the genetic system controlling the type and rate of wheat development – VRN (vernalization) at the level of an integrated system of plant organism in vivo and callus certain population of cells in vitro. The paper studies the system of allelic VRN genes, which determine the need or insensitivity Triticum aestivum L. to vernalization and predetermination process of callusogenesis by the present system in vitro. In the experiments the modern model system of nearly isogenic lines (NILs) of spring type was used. The NILs were created in genotypes of the winter varieties Myronivska 808 and Olvia. Molecular genetic analysis of allelic loci of VRN genes was carried out by PCR analysis on grains and secondary callus culture using five pairs of specific primers (Grain Gene Mass Wheat). In the course of the experiments, it was found that the genetic system controlling the wheat rate determined the frequency of callusogenesis. However, the studied genetic system did not affect the morphological characteristics of primary and secondary callus tissue. In both hexaploid wheat cultivars the maximum frequency of callusogenesis appeared to be characterized in the Vrn 2 isogenic line and the original variety, slowly developing and intensely accumulating vegetative mass in vivo. The minimal frequency of callusogenesis was determined in the VRN 1 and VRN 3 isogenic lines, characterized by the rapid development of vegetation. The callus was derived from immature wheat embryos by morphological features analysis. Calluses with low water content, mainly, amorphous, compact, transparent, white or with yellowish tint were identified. Using PCR analysis, in grains in vivo and in the callus culture in vitro the almost identical allelic status of VRN genes was revealed. In grains and callus of Vrn 1 isogenic lines of both wheat varieties the presence of a dominant gene VRN A1 and recessive VRN B1 and VRN D1 was detected, whereas in VRN 2 – a dominant gene VRN B1 and recessive VRN A1 and VRN D1. However, the dominant allele VRN D1 in the studied NILs was detected. Therefore, in varieties of grains and callus cultures all genes are represented only by recessive alleles VRN A1, VRN B1 and VRN D1. Differences were found in the callus culture of VRN 3 isogenic line of Myronivska 808 on the allelic state of theVRN B1 gene. The obtained results could be associated with genomic reconstructions during callusogenesis induction. Our studies indicate the unidirectional system of VRN genes functioning, which appears to be the main control system of type and rate of soft wheat development in the system in vivo and in vitro. This allows us to assume the role of the VRN genetic system in the determination of callusogenesis, and also the adequacy of the functioning of the system in vitro processes, determining the system of integration of plants. Thus, our study confirms that the callus tissues cells of higher plants are able to preserve the cells properties of the whole organism along with the acquisition of new specific properties. Moreover, the culture in vitro is an adequate system for the study of the plant organism properties as a system.


Achrem, M., Skuza, L., Kalinka, A., Szucko, I., Filip, E., Słominska-Walkowiak, R., Rogalska, S., 2012. Role of epigenetic mechanisms in plant response to low temperature. Acta Biol. Cracov. Bot. 54(1), 7–15.

Atramentova, L.O., Utevs’ka, O.M. (ed.), 2007. Statystychni metody v biologii’ [Statistical methods in biology]. HNU imeni V.N. Karazina, Harkiv (in Ukrainian).

Avksent’eva, O.A., Petrenko, V.A., 2009. Rol’ genotipa, sostava sredy i tipa jeksplanta v formirovanii pervichnogo kallusa izogennyh linij pshenicy [Role of the genotype, composition of medium and type of explant in formation of the first callus of wheat isogenic lines]. The Journal of V.N. Karazin Kharkiv National University. Series Bіology 771(9), 157–166 (in Russian).

Bavol, A.V., Dubrovnaja, O.V., Ljal’ko, I.I., 2008. Regeneracija rastenij iz razlichnyh tipov jeksplantov mjagkoj pshenicy [The regeneration of plants from different types of wheat explants]. Physiology and Biochemistry of Cultivated Plants 40(2), 150–156 (in Russian).

Bespalova, L.A., Koshkin, V.A., Potokina, E.K., Filibok, V.A., Matvienko, I.I., Mitrofanova, O.P., Guenkova, E.A., 2011. Photoperiod sensitivity and molecular marking of genes Ppd and Vrn in connection with breeding alternative-habit wheat varieties. Russian Agricultural Sciences 36(6), 389–392.

Chu, C.-G., Tan, C.T., Yu, G.-T., Zhong, S., Xu, S.S., Yan, L., 2011. A novel retrotransposon inserted in the dominant Vrn-B1 allele confers spring growth habit in tetraploid wheat (Triticum turgidum L.). G3. Genes, Genomes and Genetics 1(7), 637–645.

Cockram, J., Jones, H., Leigh, F., O’Sullivan, D., Powell, W., Laurie, D., Greenland, A., 2007. Control of flowering time in temperate cereals: Genes, domestication and sustainable productivity. J. Exp. Bot. 58(6), 1231–1244.

Distelfeld, A., Tranquilli, G., Li, C., Yan, L., Dubcovsky, J., 2009. Genetic and molecular characterization of the VRN2 loci in tetraploid wheat. Plant Physiol. 149(1), 245–257.

DNA extraction for PCR. Plant transformation facility, 2003. DNA Extraction from maize callus, maize leaf tissue, or soybean leaf tissue for PCR. Retrieved from

Dubcovsky, J., Loukoianov, A., Fu, D., Valarik, M., Sanchez, A., Yan, L., 2006. Effect of photoperiod on the regulation of wheat vernalization genes VRN1 and VRN2. Plant Mol. Biol. 60(4), 469–480.

Emtseva, M.V., Efremova, T.T., Arbuzova, V.S., 2012. Heading time of substitution and near-isogenic lines of common wheat with dominant alleles Vrn-B1a and Vrn-B1c. Russian Journal of Genetics: Applied Research 2(4), 304–310.

Fu, D., Szucs, P., Yan, L., Helguera, M., Skinner, J., Zitzewitz, J., Hayes, P., Dubcovsky, J., 2005. Large deletions within the first intron in VRN-1 are associated with spring growth habit in barley and wheat. Mol. Genet. Genomics 273(1), 54–65.

Golovnina, K.A., Kondratenko, E.Y., Blinov, A.G., Goncharov, N.P., 2010. Molecular characterization of vernalization loci VRN1 in wild and cultivated wheats. BMC Plant Biol. 10(1), 168.

Khotyljova, L., Kaminskaya, L., Koren, L., 2002. Influence of genetic systems of Vrn and Ppd genes on the ecological adaptation of wheat and Triticale. Biologija 4, 45–48.

Kim, D.-H., Sung, S., 2014. Genetic and epigenetic mechanisms underlying vernalization. The Arabidopsis Book 12, e0171.

Kumar, S., Sharma, V., Chaudhary, S., Tyagi, A., Mishra, P., Priyadarshini, A., Singh, A., 2012. Genetics of flowering time in bread wheat Triticum aestivum: complementary interaction between vernalization-insensitive and photoperiod-insensitive mutations imparts very early flowering habit to spring wheat. J. Genet. 91(1), 33–47.

Kunakh, V.A., 1998. Genomnaja izmenchivost’ somaticheskih kletok rastenij. Izmenchivost’ v processe dedifferencirovki i kallusoobrazovanija in vitro [Genome variability in plant somatic cells. 4. Variability in the process of dedifferentiation and callus formation in vitro]. Biopolym. Cell. 14(4), 298–319 (in Russian).

Kunakh, V.A., 2005. Biotehnologija likars’kyh roslyn. Genetychni ta fiziologo-biohimichni osnovy [Biotechnology of medicinal plants. Genetic and physiological and biochemical basis]. Logos, Kyiv (in Ukrainian).

Li, C., Distelfeld, A., Comis, A., Dubcovsky, J., 2011.Wheat flowering repressor VRN2 and promoter CO2 compete for interactions with NUCLEAR FACTOR-Y complexes. Plant J. 67(5), 763–773.

Lihenko, I.E., Stasjuk, A.I., Shherban’, A.B., Zyrjanova, A.F, Lihenko, N.I., Salina, E.A., 2014. Izuchenie allel’nogo sostava genov Vrn-1 i Ppd-1 u rannespelyh i srednerannih sortov jarovoj mjagkoj pshenicy Cibiri [Analysis of the allelic variation of the Vrn-1 and Ppd-1 genes in Siberian early and medium early varieties of spring wheat]. Vavilovskij Zhurnal Genetiki i Selekcii 18, 691–703 (in Russian).

Loukoianov, A., Yan, L., Blechl, A., Sanchez, A., Dubcovsky, J., 2005. Regulation of VRN-1 vernalization genes in normal and transgenic polyploid wheat. Plant Physiol. 138(4), 2364–2373.

Muterko, O.F., Balashova, I.A., Fayt, V.I., Syvolap, J.M., 2015. Molecular-genetic mechanisms of regulation of growth habit in wheat. Cytology and Genetics 49(1), 58–71.

Oliver, S.N., Deng, W., Casao, M.C., Trevaskis, B., 2013. Low temperatures induce rapid changes in chromatin state and transcript levels of the cereal VERNALIZATION1 gene. J. Exp. Bot. 64(8), 2413–2422.

Potokina, E.K., Koshkin, V.A., Alekseeva, E.A., Matvienko, I.I., Filobok, V.A., Bespalova, L.A., 2012. The combination of the Ppd and Vrn gene alleles determines the heading date in common wheat varieties. Russian Journal of Genetics: Applied Research 2(4), 311–318.

Shherban’, A.B., Salina, E.A., 2013. Jepigeneticheskaja reguljacija jekspressii genov jarovizacii [Epigenetic regulation of expression of vernalization genes]. Tcitologija 55(4), 234–237(in Russian).

Stelmakh, A., 1998. Genetic systems regulating flowering response in wheat. Euphytica 100(1), 359–369.

Stelpflug, S.C., Eichten, S. R., Hermanson, P. J., Springer N.M, Kaeppler, S.M., 2014. Consistent and heritable alterations of DNA methylation are induced by tissue culture in maize. Genetics 198(1), 209–218.

Stepanenko, I.L., Smirnova, O.G., Titov, I.I., 2012. A model of the gene network for flowering time regulation in winter wheat and barley. Russian Journal of Genetics: Applied Research 2(4), 319–324.

Temel, A., Gozukirmizi, N., 2013. Analysis of retrotransposition and DNA methylation in barley callus culture. Acta Biol. Hung. 64(1), 86–95.

Trevaskis, B., 2010. The central role of the VERNALIZATION 1 gene in the vernalization response of cereals. Funct. Plant Biol. 37, 479–487.

Trevaskis, B., Bagnall, D., Ellis, M., Peacock, W., Dennis, E., 2003. MADS-box genes control vernalization-induced flowering in cereals. PNAS 100(22), 13099–13104.

Yan, L., Loukoianov, A., Tranquilli, G., Helguera, M., Fahima, T., Dubcovsky, J., 2003. Positional cloning of the wheat vernalization gene VRN1. PNAS 100(10), 6263–6268.

Zhmurko, V., Avksentyeva, O., Bing, H., 2013. Influence of photoperiodic conditions on the development and content of nitrogenous compounds in the VRN NILs wheat Triticum aestivum L. Biologija 59(2), 231–240.

Zhmurko, V.V., 2010. Osobennosti projavlenija effektov genov PPD na tempy razvitija sortov i gibridov ozimoj pshenicy [Showing features of the PPD genes effects on the development pace of winter varieties and hybrids]. Faktory Jeksperimental’noj Jevoljucii Organizmov 8, 38–42 (in Russian).