Izvestiya of Saratov University.

Chemistry. Biology. Ecology

ISSN 1816-9775 (Print)
ISSN 2541-8971 (Online)

For citation:

Parhomenko A. S., Pavlenko K. S., Epifanov V. S., Efimenko S. F., Grebenyuk L. V., Skaptsov M. V. Optimization of sample preparation technique and the possibility of using different types of plant material of two species of the genus Colchicum (Colchicaceae) in flow cytometry. Izvestiya of Saratov University. Chemistry. Biology. Ecology, 2025, vol. 25, iss. 4, pp. 437-446. DOI: 10.18500/1816-9775-2025-25-4-437-446, EDN: VXQVZG

This is an open access article distributed under the terms of Creative Commons Attribution 4.0 International License (CC-BY 4.0).
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Language: 
Russian
Heading: 
Biology
Article type: 
Article
UDC: 
576.08
DOI: 
10.18500/1816-9775-2025-25-4-437-446
EDN: 
VXQVZG

Optimization of sample preparation technique and the possibility of using different types of plant material of two species of the genus Colchicum (Colchicaceae) in flow cytometry

Autors: 
Parhomenko Alena Sergeevna, Saratov State University
Pavlenko Kristina S., Saratov State University
Epifanov Vladimir S., Saratov State University
Efimenko Savelii F., Saratov State University
Grebenyuk Lyudmila V., Saratov State University
Skaptsov Mikhail V., Altai State University, South-Siberian Botanical Garden
Abstract: 

The article presents the results of testing and optimization of the sample preparation technique for fl ow cytometric analysis of two species that have not been previously studied in this aspect: Colchicum bulbocodium subsp. versicolor and C. laetum. We are using optimal LB01 buff er with added sodium thiosulfate, as internal standards we take Petroselinum crispum and Secale cereale. For the fi rst time, the DNA content in living samples of C. bulbocodium subsp. versicolor (2C = 8.049 pg) and C. laetum (2C = 5.412 pg) from natural habitats in the territory of the Russian Federation was determined. We have proven that not only living material (sprouts), but also seeds can be used as material for studying the genome size by fl ow cytometry of two species of the genus Colchicum. The main thing is to take into account their feature – the number of events corresponding to the embryo is much less than the number of endosperm cells. We do not recommend using herbarium samples of Colchicum as material for DNA content analysis, as their values were much overestimated in comparison with sprouts. The data we obtained complement and expand our understanding of the DNA level within the genus Colchicum.

Key words: 
Colchicum
plant fl ow cytometry
seeds
herbarium
seedlings
DNA content
Reference: 
  1. Оганезова Г. Г. Проблемы рода Colchicum L. Colchicum sensu lato или Colchicum sensu stricto в свете категорий прерывности и непрерывности. Ереван : НАН РА, Институт ботаники им. А. Тахтаджяна, 2019. 176 с.
  2. Vinnersten A., Manning J. A new classification of Colchicaceae // Taxon. 2007. Vol. 56, № 1. P. 171–178.
  3. Агапοва Н. Д. Числа хрοмοсοм цветкοвых растений флοры СССР: Семейства Asteraceae – Menyanthaceae / под ред. А. Л. Тахтаджяна. Л. : Наука. Ленингр. отд-ние, 1990. 509 с.
  4. Persson K. Nomenclature synopsis of the genus Colchicum (Colchicaceae) with some new species and combinations // Bot. Jahrb. Syst. 2007. Vol. 127, № 2. P. 166–242.
  5. Оганезова Г. Г. Особенности географии и направлений эволюции гистерантных и синантных видов рода Colchicum s. str. (Colchicaceae) // Takhtajania. 2011. № 1. С. 87–97.
  6. Kashin A. S., Parkhomenko A. S., Kulikova L. V., Petrova N. A., Shilova I. V., Lavrentiev M. V., Shushunov V. A. Potential Range of Bulbocodium versicolor (Ker-Gawl.) Spreng (Colchicaceae, Liliopsida) in Russia // Povolzhskiy Journal of Ecology. 2020. № 2. P. 241–247. https://doi.org/10.35885/1684-7318-2020-2-241-247
  7. Veselý P., Bureš P., Šmarda P., Pavlicek T. Genome size and DNA base composition of geophytes: The mirror of phenology and ecology? // Annals of Botany. 2012. Vol. 109, № 1. P. 65–75. https://doi.org/10.1093/aob/mcr267
  8. Šmarda P., Bureš P., Horová L., Leitch I. J., Mucina L., Pacini E., Tichý L., Grulich V., Rotreklová O. Ecological and evolutionary significance of genomic GC content diversity in monocots // Proceedings of the National Academy of Sciences. 2014. Vol. 111, № 39. P. 4096–4102. https://doi.org/10.1073/pnas.1321152111
  9. Bou Dagher-Kharrat M., Abdel-Samad N., Douaihy B., Bourge M., Fridlender A., Siljak-Yakovlev S., Brown S. C. Nuclear DNA C-values for biodiversity screening: Case of the Lebanese flora // Plant Biosystems. 2013. Vol. 147, № 4. P. 1228–1237. https://doi.org/10.1080/11263504.2013.861530
  10. Fridlender A., Brown S., Verlaque R., Crosnier M. T., Pech N. Cytometric determination of genome size in Colchicum species (Liliales, Colchicaceae) of the western Mediterranean area // Plant Cell Reports. 2002. Vol. 21, № 4. P. 347–352. https://doi.org/10.1007/s00299-002-0522-4
  11. Николаева M. Г., Разумова M. В., Гладкова В. Н. Справочник по проращиванию покоящихся семян. Л. : Наука. Ленингр. отд-ние, 1985. 506 с.
  12. Skaptsov M. V., Kutsev M. G., Smirnov S. V., Vaganov A. V., Uvarova O. V., Shmakov A. I. Standards in plant flow cytometry: An overview, polymorphism and linearity issues // Turczaninowia. 2024. Vol. 27, № 2. P. 86–104. https://doi.org/10.14258/turczaninowia.27.2.10
  13. Грибок Н. А., Власова Т. М., Матюшина М. В., Курченко В. П. Содержание вторичных метаболитов у представителей рода Colchicum L., интродуцированных в условиях Беларуси // Тр. Белорус. гос. ун-та. Сер.: Физиол., биохим. и молекуляр. основы функционирования биосистем. 2010. Т. 4, ч. 2 : Инновац. биотехнологии XXI в. С. 129–137.
  14. Doležel J., Sgorbati S., Lacretti S. Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants // Physiol. Plant. 1992. № 85. P. 625–631.
  15. Obermayer R., Leitch I. J., Hanson L., Bennett M. D. Nuclear DNA C-values in 30 species double the familial representation in pteridophytes // Ann. Bot. 2002. Vol. 90, № 2. P. 209–217. https://doi.org/10.1093/aob/mcf167
  16. Doležel J., Greilhuber J., Lucretti S., Meister A., Lysák M., Nardi L. Plant genome size estimation by flow cytometry: Inter-laboratory comparison // Ann. Bot. 1998. Vol. 82. P. 17–26 https://doi.org/10.1006/anbo.1998.0730
  17. Levan A. Bulbocodium to the c-mitotic action of colchicine // Cyto-genetic laboratory of the Swedish seed association. 1947. Vol. 33, № 4. P. 552–566. https://doi.org/10.1111/j.1601-5223.1947.tb02821.x
  18. Кунах В. А. Пластичность генома соматических клеток и адаптивность растений // Молекулярная и прикладная генетика. 2011. № 12. С. 7–12.
  19. Matzk F., Meister A., Schubert I. An efficient screen for reproductive pathways using mature seeds of monocots and dicots // Plant J. 2000. Vol. 21, №1. P. 97–108. https://doi.org/10.1046/j.1365-313x.2000.00647.x
  20. Servick S., Visger C. J., Gitzendanner M. A., Soltis P. S., Soltis D. E. Population genetic population, geographic structure, and multiple origins of autopolyploidy in Galax Urceolata // Am. J. Bot. 2015. № 102. P. 973–982. https://doi.org/10.3732/ajb.1400554
  21. Suda J., Trávnícek P. Reliable DNA ploidy determination in dehydrated tissues of vascular plants by DAPI flow cytometry new prospects for plant research // Cytometry A. 2006. Vol. 4. P. 273–80. https://doi.org/10.1002/cyto.a.20253
  22. Viruel J., Conejero M., Hidalgo O., Pokorny L., Powell R. F., Forest F., Kantar M. B., Soto Gomez M., Graham S. W., Gravendeel B., Wilkin P., Leitch I. J. A Target Capture-Based Method to Estimate Ploidy From Herbarium Specimens // Frontiers in Plant Science. 2019. Vol. 10. URL: https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls... (дата обращения: 15.05.2025).
  23. Skaptsov M. V., Vaganov A. V., Kechaykin A. A., Kut sev M. G., Smirnov S. V., Dorofeev V. I., BorodinaGra bovskaya A. E., Seregin A. P., Sinitsina T. A., Friesen N., Zhang X., Shmakov A. I. The cytotypes variability of the complex Selaginella sanguinolenta s. l // Turczaninowia. 2020. № 23. P. 5–14. https://doi.org/10.14258/turczaninowia.23.2.1
  24. Bainard J. D., Husband B. C., Baldwin S. J., Fazekas A. J., Gregory T. R., Newmaster S. G., Kron P. The effects of rapid desiccation on estimates of plant genome size // Chromosome Res. 2011. № 19. P. 825–842. https://doi.org/10.1007/s10577-011-9232-5
  25. Wang G., Yang Y. The effects of fresh and rapid desiccated tissue on estimates of Ophiopogoneae genome size // Plant Divers. 2016. № 38. P. 190–193. https://doi.org/10.1016/j.pld.2016.08.001
  26. Tomaszewska P., Pellny T. K., Hernández L. M., Mitchell R. A. C., Castiblanco V., de Vega J. J., Schwarzacher T., Heslop-Harrison P. Flow Cytometry-Based Determination of Ploidy from Dried Leaf Specimens in Genomically Complex Collections of the Tropical Forage Grass Urochloa s. l. // Genes. 2021. № 12. URL: https://pubmed.ncbi.nlm.nih.gov/34201593/ (дата обращения: 15.05.2025).
  27. Staats M., Cuenca A., Richardson J. E., Vrielink-van Ginkel R., Petersen G., Seberg O., Bakker T. F. DNA Damage in Plant Herbarium Tissue // PLoS ONE. 2011. Vol. 12, № 6. P. 1–9. https://doi.org/10.1371/journal.pone.0028448
  28. Darzynkiewicz Z., Traganos F., Kapuscinski J., Staiano-Coico L., Melamed M. R. Accessibility of DNA in situ to various fluorochromes: Relationship to chromatin changes during erythroid differentiation of Friend leukemia cells // Cytometry A. 1984. Vol. 5, № 4. P. 355–363. https://doi.org/10.1002/cyto.990050411
Received: 
15.08.2025
Accepted: 
05.09.2025
Published: 
25.12.2025
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