In order to establish the suitability of previously developed micropropagation protocol to get genetically uniform plants of D. antarctica the genetic variation of the plants in the process of propagation and maintenance in vitro was examined using PCR analysis and cytogenetic analysis. The clonal progeny was evaluated for the plants that differed by the molecular-genetic markers and cytogenetic characteristics. Compаrative analysis of plants at the initial stages (1—6 passages) of propagation and after a long-term maintenance in vitro (24—26 passages and more) does not reveal genetic differentiation among the clones of common origin and their original genotype by ISSR-markers. Analysis of long-term cultured plants (49—79 passages) also showed maintenance of initial cytogenetic characteristics in the micropropagated plants. In general, the results prove that the developed technique provides the preservation of genetic chаrаcteristics of D. antarctica during long-term maintenance in vitro and can be used to produce genetically uniform plant material.
Keywords: Deschampsia antarctica, micropropagation, genome variation, PCR-analysis, cytogenetic analysis
Full text and supplemented materials
Free full text: PDFReferences
1. Zagrichuk, O.M., Drobik, N.M. & Kozeretska, I.A. (2011/2012). Introduction to the culture of in vitro Deschampsia antarctica Desv. (Poaceae) from two areas of the Coastal Antarctic. Ukrayinskiy antarktichniy zhurnal, 10-11, pp. 289-295 [in Ukrainian].
2. Kunakh, V.A. (2005). Biotechnology of Medicinal Plants. Genetic and physiological and biochemical bases. Kyiv: Logos [in Ukrainian].
3. Kunakh, V.A. (2010). Extras or B-chromosomes of plants. Origin and biological significance. Visnyk ukrayinskogo tovaristva genetikiv i selektioneriv, 8, No. 1, pp. 99-139 [in Ukrainian].
4. Ozheredova, I.R., Parnikoza, I.Yu. & Poronnik, O.O. (2015). The mechanism of adaptation of Antarctic vascular plants to abiotic environmental factors. Cytology and Genetics, 49 (2), pp. 72-79 [in Ukrainian]. https://doi.org/10.3103/S0095452715020085
5. Parnykoza, I.Yu., Abakumov, E.V. & Dykiy, I.V. (2014). Ornithogenic localities of Deschampsia antarctica in the region of the Argentine Islands (Coastal Antarctica). Ruskiy ornytologicheskiy zhurnal, 23(1056), pp. 3095-3107 [in Russian].
6. Soloveva, L.V. & Plehanova, N.M. (1992). About additional chromosomes in honeysuckle. Cytology and Genetics, 26(3), pp. 21-25 [in Russian].
7. Tsytlenok, S.I. & Pulkyna, S.V. (1991). Chromosome polymorphism Crepis sibirica (Asteraceae). Botanical Journal, 76 (11), pp. 1538-1544 [in Russian].
8. Alberdi, M., Bravo, L.A., Gutierrez ,A. & Gidekel, M. (2002). Ecophysiology of Antarctic vascular plants. Physiologia Plantarum, 115 (4), pp. 479-486. https://doi.org/10.1034/j.1399-3054.2002.1150401.x
9. Amosova, A.V., Bolsheva, N.L., Samatadze, T.E., Twardovska, M.O., Zoshchuk, S.A., Andreev, I.O., Badaeva, E.D., Kunakh, V.A. & Muravenko, O.V. (2015). Molecular cytogenetic analysis of Deschampsia antarctica Desv. (Poaceae), Maritime Antarctic. PLoS One, 10 (9), e0138878. https://doi.org/10.1371/journal.pone.0138878
10. Bhatia, R., Singh, K.P., Sharma, T.R. & Jhang, T. (2011). Evaluation of the genetic fidelity of in vitro-propagated gerbera (Gerbera jamesonii Bolus) using DNA-based markers. Plant Cell, Tissue Organ Cult., 104, pp. 131-135. https://doi.org/10.1007/s11240-010-9806-5
11. Caetano-Anolles, G. (1999). High genome-wide mutation rates in vegetatively propagated bermudagrass. Molecular Ecology, No. 8, pp. 1211-1221. https://doi.org/10.1046/j.1365-294x.1999.00702.x
12. Chiavarino, A.M., Rosato, M., Rosi, P., Poggio, L. & Narango, C.A. (1998). Localization of the genes controlling B chromosome transmission rate in maize (Zea mays ssp. mays, Poaceae). American Journal of Botany, 85 (11), pp.1581-1585. https://doi.org/10.2307/2446484
13. Cuba, M., Gutierrez-Moraga, A., Butendieck, B. & Giedekel, M. (2015). Micropropagation of Deschampsia antarctica — a frost-resistant Antarctic plant. Antarct. Sci., 17 (1), pp. 69-70. https://doi.org/10.1017/S0954102005002440
14. Doyle, J.J. & Doyle, J.L. (1987). A rapid DNA isolation of fresh leaf tissue. Phytochemical Bulletin, No. 19, pp.11-15.
15. Edwards, J.A. (1972). Studies in Colobanthus quitensis (Kunth) Bartl. and Deschampsia antarctica Desv. V. Distribution, ecology and vegetative performance on Signy Island. British. Antarct. Surv. B., No. 28, pp. 11-28.
16. Gajdosova, A., Ostrolucka, M.G., Libiakiva, G., Ondruskova, E. & Simala, D. (2006). Microclonal propagation of Vaccinium sp. and Rubus sp. and detection of genetic variability in culture in vitro. Journal of Fruit and Ornamental Plant Research, 14 (1), pp.103-119.
17. Gantait, S., Mandal, N., Bhattacharyya, S. & Das, P.K. (2010). Determination of genetic integrity in long-term micropropogated plantlets of Allium ampeloprasum L. using ISSR markers. Biotechnology, 9 (2), pp. 218-223. https://doi.org/10.3923/biotech.2010.218.223
18. Joshi, P. & Dhawan, V. (2007). Assessment of genetic fidelity of micropropagated Swertia chirayita plantlets by ISSR marker assay. Biologia Plantarum, 51 (1), pp. 22-26. https://doi.org/10.1007/s10535-007-0005-0
19. Lespinasse, R. (1987). Genese des chromosomes B el leur avenir evolutif. Actes Colloq. biol. populat. Lyon, pp. 427-432.
20. Levin, D.A., Palestis, B.G., Jones, R.N. & Trivers, R. (2005). Phyletic hot spots for B chromosomes in angiosperms. Evolution, No. 59, pp. 962-969. https://doi.org/10.1111/j.0014-3820.2005.tb01035.x
21. Mosula, M.Z., Andreev, I.O., Bublyk, O.M. & Mel'nyk, V. (2015). Molecular markers to assess genetic diversity of Gentiana lutea L. from the Ukrainian Carpathians. Plant Genetic Resources, No. 13, pp. 266-273. https://doi.org/10.1017/S147926211400104X
22. Mozafari, A.A., Vafaee, Y. & Karami, E. (2015). In vitro propagation and conservation of Satureja avromanica Maroofi — an indigenous threatened medicinal plant of Iran. Physiology and Molecular Biology of Plants, 21 (3), pp. 433-439. https://doi.org/10.1007/s12298-015-0313-3
23. Patra, N.K., Srivastava, H.K. & Shauhan, S.P. (1988). B chromosomes in spontaneous and induced intercellular chromosome migration of Papaver somniferum. Indian. J. Genet. Plant Breed., 48 (1), pp. 31-42.
24. Ray, T., Dutta, I., Saha, P. & Das, S. (2006). Genetic stability of three economically important micropropagated banana (Musa spp.) cultivars of lower Indo-Gangetic plains, as assessed by RAPD and ISSR markers. Plant Cell Tissue and Organ Culture, 85, pp.11-21. https://doi.org/10.1007/s11240-005-9044-4
25. Saralva, L.S. & de Carvalho, C.B. (1993). Genetic evidence of an internal deletion induced by B chromosomes in maize (Zea mays L.). Rev. Bras. Genet., 6 (1), pp. 107-113.
26. Schluter, P.M. & Harris, S.A. Analysis of multilocus fingerprinting data sets containing missing data. Molecular Ecology Notes, 6 (2), pp. 569-572. https://doi.org/10.1111/j.1471-8286.2006.01225.x
27. Soni, M. & Kaur, R. (2014). Rapid in vitro propagation, conservation and analysis of genetic stability of Viola pilosa. Physiology and Molecular Biology of Plants, 20 (1), pp.95-101. https://doi.org/10.1007/s12298-013-0200-8
28. Verma, N., Koche, V., Tiwari, K.L. & Mishra, S.K. (2010). Random amplified polymorphic DNA analysis detects variation in a micropropagated clone of Trichodesma indicum (L.) R. Br. African Journal of Biotechnology, 9 (28), pp.4322-4325.