PLOIDY LEVEL AND RELATIVE NUCLEAR DNA CONTENT IN THE PLANT CELL AND TISSUE CULTURE IN VITRO

M. V. Skaptsov, M. A. Krasnoborodkina, M. G. Kutsev, S. V. Smirnov, A. I. Shmakov, A. V. Matsyura

Abstract


We presented results of variations in the ploidy level and the genome size of the R. acetosa regenerants. These regenerants was obtained by indirect and direct morphogenesis in in vitro culture. Explants were prepared from seedlings on the three-leaf stage of plant development. More than 100 explants were used to stimulate the indirect and direct morphogenesis. Mesophilic explants were cultured on the MS nutrient medium containing auxin to callus proliferation (2 mg/L NAA, 1 mg/L BA). Cultivation of the callus was maintained for 4 weeks followed by an indirect morphogenes. Indirect morphogenesis stimulated on the MS medium with cytokinin and gibberellic acid predominance (0.5 mg/L BA, 0.2 mg/L GA3). Direct stimulate morphogenesis from the apical meristem of seedlings on nutrient media with a predominance of cytokinins (1 mg/L BA, 0.25 mg/L NAA). Rhizogenesis have stimulated by transferring of the regenerants to the ½MS medium supplemented with 0.2 mg/L of NAA. Research of a ploidy level and genome size was performed by flow cytometry used propidium iodide staining with Vicia faba cv “Innovec” (2C=26.90 pg) as internal DNA standard. We calculated the relative DNA content (2C) for R. acetosa equal to 6,98 pg. Cytogenetical analisis showed that the maximum genome size variation recorded for regenerants obtained through the indirect morphogenesis. Variations in the genome size of the regenerants obtained by direct morphogenesis deviates from the control group to 0.30 pg (2С=7.28 pg) and after indirect morphogenesis to 1.04 pg (2С=8.2 pg). Cytogenetical analysis of the regenerated plants showed the presence of different somatic chromosome numbers ranging from 2n = 14 to 2n = 28. The relative DNA content of tetraploid forms was 11.87 pg. In our study was shown, that the most effective method of plant conservation in the in vitro culture is a direct morphogenesis. Analysis of the relative nuclear DNA content and chromosome counts of regenerants obtained by indirect morphogenesis from the callus cultures showed significant variations in the DNA content, as well as the appearance of polyploid forms. Therefore, long-term cultivation of callus cultures increases the probability of genomic aberrations, which reduces the stability of the plant genome.


Keywords


R. Acetosa; callus; flow cytometry; genome size; polyploidy; somaclonal variation

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References


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Kaeppler, S.M., Kaeppler, H.F., & Rhee, Y. (2000). Epigenetic aspects of somaclonal variation in plants. Plant Mol Biol, 43, 179–188.

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Larkin, P.J., & Scowcroft, W.R. (1981). Somaclonal variation: a novel source of variability from cell cultures for plant improvement. Theor Appl Genet, 60, 197–214.

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Miler, N., & Zalewska, M. (2014). Somaclonal variation of Chrysanthemum propagated in vitro from different explants types. Acta Scientiarum Polonorum Hortorum Cultus, 13(2), 69–82.

Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol, 15(13), 473–497.

Orbović, V., Ćalović, M., Viloria, Z., Nielsen, B., Gmitter, F.G., Castle, W.S., & Grosser, J.W. (2008). Analysis of genetic variability in various tissue culture-derived lemon plant populations using RAPD and flow cytometry. Euphytica, 161(3), 329–335.

Otto, F. (1992). Preparation and staining of cells for high-resolution DNA analysis. In A. Radbruch (Eds.). Flow cytometry and cell sorting (pp. 101–104). Berlin: Springer Verlag.

Palomino, G., Doležel, J., Cid, R., Brunner, I., Mendez, I., & Rubluo, A. (1999). Nuclear genome stability of Mammillaria san-angelensis (Cactaceae) regenerants induced by auxins in long-term in vitro culture. Plant Science, 141, 191–200.

Peakall, R., & Smouse, P.E. (2012). GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research an update. Bioinformatics, 28, 2537–2539.

Pfosser, M., Amon, A., Lelley, T., & Heberle-Bors, E. (1995). Evaluation of sensitivity of flow cytometry in detecting aneuploidy in wheat using disomic and ditelosomic wheat-rye addition lines. Cytometry, 21(4), 387–393.

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Sun, S., Zhong, J., Li S., & Wang, X. (2013). Tissue culture-induced somaclonal variation of decreased pollen viability in torenia (Torenia fournieri Lind.). Botanical Studies, 54 (36).

Tanaka, R. (1959). On the speciation of karyotype in diploid and tetraploid species of Chrysanthemum boreale (2n=18). Journal of Science of Hiroshima University Series B Division 2, 9, 1–16.

Trader, B.W., Gruszewski, H.H., Scoggins, H.L., & Veilleux, R.E. (2006). Somaclonal variation of Coreopsis regenerated from leaf explants. Horticultural Science, 41(3), 749–752.

Viehmannova, I., Bortlova, Z., Vitamvas, B., Cepkova, P.H., Eliasova, K., Svobodova, E. & Travnickova, M. (2014).

Assessment of somaclonal variation in somatic embryo-derived plants of yacon [Smallanthus sonchifolius (Poepp. and Endl.) H. Robinson] using inter simple sequence repeat analysis and flow cytometry. Electronic Journal of Biotechnology, 17(2), 102–106.

Yang, M., & Loh, C. S. (2004). Systemic endopolyploidy in Spathoglottis plicata (Orchidaceae) development. BMC Cell Biology, 5(33).




DOI: http://dx.doi.org/10.15421/201667

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