Genetic variability of tobacco plants regenerated from cell lines resistant to heavy metal ions
DOI:
https://doi.org/10.29038/NCBio.24.2-2Keywords:
Nicotiana tabacum (L.), cell selection, heavy metal ions, cytometric analysis, polyploidy.Abstract
One of the main elements of cytological observations carried out at all stages of creating new forms of plants is the determination of ploidy, both of the crossing components and of the resulting hybrids, varieties, and variants. The traditional method of determining ploidy by counting chromosomes on pressurized or permanent preparations under a microscope is laborious, and other methods of determining ploidy by the size of oysters, pollen or the number of chloroplasts in epidermal stomatal cells are not very accurate, especially if the ploidy of the samples does not differ significantly.
The most reliable method for determining plant ploidy is the cytophotometric method of ploidy determination based on changes in the amount of DNA in the cell. The essence of flow cytometry (FCM) is that the fluorochrome light pulse recorded by a flow cytometer is proportional to the amount of DNA of each individual cell nucleus, in turn, the amount of DNA of the interphase nucleus reflects the ploidy of the latter.
Data on the analysis of fertility in experimental plants are presented. The genomes of tobacco (Nicotiana tabacum L.) regenerants obtained from cell lines resistant to barium, cadmium, and vanadium ions were studied by cytometric analysis. It was found that the regenerant obtained from the cadmium (Cd2+) resistant line is a triploid. The regenerants obtained from cell lines resistant to barium (Ba2+) and vanadium ions are mixoploids. Control tobacco plants have a diploid genome. Regenerants retain the phenotypic characteristics of resistant cell lines. In our opinion, these characteristics are probably related to the phenomenon of polyploidy. However, other types of mutations in the studied tobacco plant variants are not excluded.
References
1. Roik, M.V.; Kovalchuk, N.S.; Aleksiychuk, L.V. Analiz minlivosti rivnya ploidnosty genomu vihidnih selekciynih materialiv cukrovih buryakiv z vikoristannyam tehnologii analizatora ploidnosti «Partec». Кyiv, 2006, 39с.
2. Murashige, T.; Skoog, F. A revised medium for rapid growth and biossays with tobacco tissue culture. Physiology Plants. 1962, 15(13), 473-497
3. Tores – Silva, G.; Matos, E.M.; Correla, L.F.; Fortini, E.A.; Soares, S.S.; Batista, D.S.; Otoni, C.G.; Azevedo, A.A.; Viccini, L.F.; Koehler, A.D.; Resende, S.V.; Specht, C.D.; Otoni, W.C. Anatomy, flow cytometry, and x-ray tomography reveal tissue organization and ploidy distribution in long-term in vitro cultures of Melocactus species. Frontuers Plant Science. 2020, 11(1314), 1-13 https://doi.org/10.3389/fpls.2020.01314
4. Gupta, N.; Mittal, A.; Dadu, T.; Choudhary, D.; Handoo, A. Flow cytometric DNA ploidy analysis in haemato-limphoid neoplasms: an analysis of 132 cases. International Journal of Hematology-Oncology and Stem Cell Research. 2022, 16(1), 34-45
5. Zhao, H.; Albino, A.P.; Jorgensen, E.; Traganos, F.; Darzynkiewicz, Z. DNA damage response induced by tobacco smoke in normal muman bronchial epithelial and A549 Pulmonary adenocarcinoma cells assessed by laser scanning cytometry. International Society for Advancement of Cytometry. Cytometry, Part A. 2009, 75A, 840-847 https://doi.org/10.1002/cyto.a.20778
6. Widoretno, W.; Azriningsih, R.; Sukmadjaja, D.; Rosyidah, M. In vitro induction and identification of poliploidAmorphophallusmuelleri Blume plants by colchicine treatment. Agrivita Journal Science. 2023, 45(1), 87-97 https://doi.org/10.17503/agrivita.v45i1.3992
7. Sliwinska, E. Flow cytometry – a modern method for exploring genome size and nuclear DNA synthesis in horticural and medicinal plant species. Folia Horticulturae. 2018, 30(1), 103-128 https://doi.org/10.2478/fhort-2018-0011
8. Sjahril, R.; Kasmiati, R.M.; Jamaluddin, I.; Panga, N.J. Flow cytometry analisis on colchicine induced poliploid of Katokkon peppers (Capsicum chinense Jacq.). IOP Conf. Ser.: Earth Environmental Science. 2021, 807(032024), 1-6 https://doi.org/10.1088/1755-1315/807/3/032024
9. Fomicheva, M.; Domblides, E. Mastering DNA content estimation by flow cytometry as an efficient tool for plant breeding and biodiversity research. Method and protocols. 2023, 6(18), 1-13 https://doi.org/10.3390/mps6010018
10. Galbaraith, D.; Loureiro, J.; Antoniadi, I.; Bainard, J.; Bureš, P.; Càpal, P.; Castro, M.; Castro, S.; Doležel, J.; Giorgi, D.; Husband, B.C.; Kolaŕ, F.; Kouteckŷ, P.; Kron, P.; Leitch, I.J.; Lung, K.; Lopes, S.; Lućanovà, M.; Lucretti, S.; Ma, W.; Melzer, S.; Molnàr, I.; Novàk, O.; Poulton, N.; Skalickŷ, V.; Sliwinska, E.; Šmarda, P.; Smith, T.W.; Sun, G.; Talhinhas, P.; Tàrnok, A.; Temsch, E.M.; Tràvniček, P.; Urfus, T. Best practices in plant cytometry. International Society for Advancement of Cytometry. 2020, 7(4), 311-317 https://doi.org/10.1002/cyto.a.24295
11. Metcalfe, C.J.; Li, J.; Doležel, J.; Piperidis, N.; Aitken, K.S. Flow cytometric characterization of the complex polyploidy genome of Saccharum officinarum and modern sugarcane cultivars. Scientific reports. 2019, 9(19362), 1-12 https://doi.org/10.1038/s41598-019-55652-3
12. Augustine, S.M.; Cherian, A.V.; Seiling, K.; Fiore, S.D.; Raven, N.; Commandeur, U.; Schillberg, S. Targeted mutagenesis in Nicotiana tabacum ADF gene using shocwave – mediated ribonucleoprotein delivery increases osmotic stress tolerance. Physiologia Plantarum. 2021, 173, 993-1007. https://doi.org/10.1111/ppl.13499
13. Huang, T.-K.; Armstrong, B.; Schindele, P.; Puchta, H. Efficient gene targetingn in Nicotiana tabacum using CRISPR/SaCas9 and temperature tolerant LbCas12a. Plant Biotechnology Journal. 2021, 19, 1314-1324 https://doi.org/10.1111/ppi.13546
14. Lewis, R.S.; Drake-Stowe, K.E.; Heim, C.; Steede, T.; Smith, W.; Dewey, R.E. Genetic and agronomic analisis of tobacco genotypes exhibiting reduced nicotine accumulation due to induced mutations in Berberine Bridge Like (BBL) genes. Frontiers in Plant Science. 2020, 11(368), 1-14 https://doi.org/10.33.89/fpls.2020.00368
15. Pikalo, S.V.; Dubrovna, O.V. Riven' ploidnosti roslyn – regenerantiv triticale, otrymanyh shlyahom selekcii in vitro na stiykist' do abiotychnyh stresiv. Factory eksperimentalnoy evolucii organizmiv. 2018, 22, 305 – 310. https://doi.org/10.7124/FEEO.V22.966
16. Ignatiev, O.M.; Panyuta O.I.;Oparina, T.P.; Prutyan, T.L. Diyalnist' zstosuvannya protochnoyi cytometrii na osnovy analizu zmin u zagalnomu analizi krovy, hvoryh na на COVID – 19. Naukoviy visnik Uzgorodskogo universitetu, seriya «Medicina». 2022, 2(66), 62 – 66. https://doi.org/10.32782/2415-8127.2022.66.11
17. Lysenko, S.A. Protochna DNK – cytometrya v ocincy paraneoplastychnogo revmatologichnogo syndromy u hvoryh na rak legen'. – Ukrainskiy naukovo – medychniy molodyzhniy zhurnal. Klinichna medicyna. 2013, 4, 32 – 36.
18. Mykytyuk, O.Y. Protochna cytometriya: fiziologichny osnovi ta praktychne zasosuvannya u mediciny i biologi. Visnik problem biologii i mediciny. 2015, Вип.2, 1(118), 214 - 217
19. Paes de Melo, B.; Carpinetti de Avelar, P.; Fraga, O.T.; Rodrigues – Silva, P.L.; Sartory-Fioresi, V.; Fernando de Camargos, L.; Ferreira de Silva, M.F. Abiotic stresses in plants their markers: a responses and programmed cell death mechanisms. Journals Pants. 2022, 11(9), 1100 – 1119 https://doi.org/10.3390/plants11091100
20. Zhang, Y.; Xu, J.; Li, R.; Ge, Y.; Li, Y.; Li, R. Plant response to abiotic stress: mechanisms and strategies. International Journal Molecular Science. 2023, 24(13), 10915 https://doi.org/10.3390/ijms241310915
21. Sergeeva, L.E.; Mykhalska, S.I. Cell selection with heavy metal ions for obtaining salt tolerant plant cell cultures. Fiziology plants and genetics. 2019. 51(4), 315 – 323 https://doi.org/frg2-019.04.315
22. Sergeeva, L.; Bronnikova, L. Cell selectiom with bareium ions for obtaining genetically modified tobacco. Вісник Черкаського університету Серія біологія. 2020, 1, 71 – 78 https://doi.org/10.31651.2076-5835-2018-1-2020-1-71-78
23. Sergeeva, L.E. Nitrate reductase activity in biotechnology tobacco plants under enzyme inhibitors action. Fiziology plants and genetics. 2017, 49(2), 129 – 133 https://doi.org/10.15407/frg2017.02.129
Downloads
Published
Issue
Section
License
Copyright (c) 2025 Bronnikova L.I., Zaitseva I.O.

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.