Assessment of Genetic Stability of Propagated Plants of Alyssum caricum Using Flow Cytometry

Year 2023, Volume: 11 Issue: 2, 246 - 252, 28.12.2023

Nurşen Çördük , Gülru Yücel

https://doi.org/10.33202/comuagri.1367786

Abstract

This study aimed to investigate the nuclear DNA content of A. caricum using in vitro-grown plants and to analyse the genetic stability of in vitro-propagated plantlets through flow cytometry. The process involved germinating seeds on Murashige and Skoog medium with 3% sucrose and 0.7% phytoagar. The germinated seedlings were then cultured in vitro in a controlled environment. For the in vitro propagation experiment, nodal explants were cultured on three shoot induction media for 3-4 weeks. Subsequently, the adventitious shoots were transferred to the MS medium with the same formulations as the shoot induction medium to facilitate shoot propagation. The propagated adventitious shoots were then moved to full-strength MS medium with 3% sucrose and 0.7% phytoagar, without any plant growth regulators, to induce root development. The nuclear DNA content was estimated using flow cytometry for both the in vitro germinated seedlings and the propagated plantlets. The analyses revealed that A. caricum had 1.75 pg/2C±0.01 mean nuclear DNA content. Furthermore, the propagated plants exhibited similar nuclear DNA content. These results suggest that the analysed individuals were genetically stable. However, to obtain more comprehensive information about A. caricum, additional studies such as chromosome counting, genetic analyses are necessary.

Keywords

A. caricum, In vitro propagation, Nuclear DNA Content, Genetic stability, Flow cytometry

Project Number

FBA-2018-1439

Assessment of Genetic Stability of Propagated Plants of Alyssum caricum Using Flow Cytometry

Abstract

Bu çalışmada, flow sitometri analizi ile in vitro yetiştirilmiş A. caricum bitkilerinin çekirdek DNA içeriğinin belirlenmesi, ayrıca in vitro çoğaltılan bitkilerin genetik stabilitelerinin değerlendirilmesi amaçlanmıştır. Bitkinin tohumları, 3% sukroz ve 0.7% fitoagar içeren Murashige ve Skoog besin ortamında çimlendirilmiştir. Çimlenen fideler kontrollü bir ortamda in vitro olarak büyütülmüştür. In vitro çoğaltım için nodal eksplantlar üç farklı sürgün indüksiyon ortamında 3-4 hafta kültüre alınmıştır. Adventif sürgünler sürgün indüksiyon ortamı ile aynı formülasyonlara sahip MS besin ortamına çoğaltılmaları için aktarılmıştır. Çoğaltılan adventif sürgünler, daha sonra bitki büyüme düzenleyicileri içermeyen MS ortamına aktarılmıştır. In vitro yetiştirilen bitkiler ve in vitro çoğaltılan bitkiler flow sitometri ile analiz edilmiştir. Yapılan flow sitometri analizleri, in vitro yetiştirilen bitkilerin 1.75 pg/2C±0.01 DNA içeriğine sahip olduğunu göstermiştir. Sonuçlar, çoğaltılan bitkilerin tohumdan çimlenen bitkiler ile benzer DNA içeriğine sahip olduğunu göstermiştir. Bu sonuçlar, analiz edilen bireylerin genetik olarak stabil olduğunu önermektedir. Bununla birlikte, A. caricum hakkında daha kapsamlı bilgilere ulaşmak için ek çalışmalar gerekmektedir.

Keywords

A. caricum, In vitro çoğaltım, Çekirdek DNA içeriği, Genetik stabilite, Flow sitometri.

Ethical Statement

There are no ethical issues after the publication of this manuscript.

Supporting Institution

Çanakkale Onsekiz Mart University The Scientific Research Coordination Unit

Project Number

FBA-2018-1439

References

• Adıgüzel, N., Reeves, R.D., 2002. A new nickel-accumulating species of Alyssum (Cruciferae) from Western Turkey. Edinb. J. Bot. 59: 215–219.
• Adıgüzel, N., Reeves, R.D., 2012.Important serpentine areas of Turkey and distribution patterns of serpentine endemics and nickel accumulators. Bocconea. 24: 7-17.
• Arslan, A., 2019. Investigation of pollen, seed, fruit morphology and antimicrobial and antibiofilm activity of some Alyssum L. species in Anatolian flora. Master Thesis, Bartın University, Graduate School of Natural and Applied Sciences.
• Aytaç, Z., Duman, H., 2000. A new species of Alyssum L. (Cruciferae) from Central Anatolia. Isr. J. Plant Sci. 48: 317–320.
• Babaoğlu, S., Bani, B., Açık, L., Adıgüzel, N., 2006. Taxonomic relations among some Turkish serpentine endemic Alyssum (Brassicaceae). Plant, fungal and habitat diversity investigation and conservation Proceedings of IV BBC – Sofia. 192-195.
• Bayramoglu, G., Adiguzel, N., Ersoy, G. Yilmaz M., Arica M. Y., 2013. Removal of textile dyes from aqueous solution using amine-modified plant biomass of A. caricum: equilibrium and kinetic studies. Water Air Soil Pollut. 224: 1640.
• Çatav, Ş.S., Küçükakyüz, K., Tavşanoğlu, Ç., Akbaş K., 2015. Effects of aqueous smoke and nitrate treatments on germination of 12 Eastern Mediterranean Basin plants. Annales Botanici Fennici. 52(1–2): 93-100.
• Chinnusamy, V., Zhu, J.K., 2009. Epigenetic regulation of stress responses in plants. Curr Opin Plant Biol. 12:133-9.
• Çördük, N., Cambaz, E., Yılmaz, A., Yılmaz, Ö., 2023. Effect of MS nutrient salts and plant growth regulators on the in vitro propagation of Alyssum caricum T.R.Dudley & Hub.-Mor. (Brassicaceae), an endemic species in the Flora of Türkiye. Ed. Deniz NG. Science and Mathematics in a Globalizing World, Duvar Publishing. 1-34.
• Çördük, N., Yücel, G., Akıncı, N, Tuna, M., 2017. Assessment of the genetic stability of indirect shoot organogenesis-derived plantlets of Digitalis trojana Ivanina by flow cytometry and cytological analyses. Journal of Tekirdag Agricultural Faculty. 14 (1): 69-76.
• Çördük, N., Yücel, G., Akıncı, N., Tuna, M., Esen, O., 2018. In vitro propagation of Silene bolanthoides Quézel, Contandr. & Pamukç. and assessment of genetic stability by flow cytometry. Archives of Biological Sciences. 70(1): 141-148.
• Doležel, J., Greilhuber, J., Suda, J., 2007. Estimation of nuclear DNA content in plants using flow cytometry. Nature Protocols. 2: 2233–2244.
• Dudley, T.R., 1964. Synopsis of the genus Alyssum. J. Arnold Arbor. 45(3): 358– 373.
• Dudley, T.R., 1965. Alyssum L. in Flora of Turkey and the East Aegean Islands, Ed.: Davis, P.H., Edinburgh: Edinburgh University Press., 1: 362–409.
• Ekim, T., Koyuncu, M., Vural, M., Duman, H., Aytac, Z., Adıguzel, N., 2000. Red data book of Turkish plants (Pteridophyta and Spermatophyta). Ankara: Foundation for Turkish Nature Conservation and Van Centinential University Press; 79 p.
• Escobedo-Gracia-Medrano, R.M., Burgos-Tan, M.J., Ku-Cauich, J.R., Quiroz-Moreno, A.,2018. Using flow cytometry analysis in plant tissue culture derived plants. In: Loyola-Vargas, V., Ochoa-Alejo, N. (eds) Plant Cell Culture Protocols. Methods in Molecular Biology, vol 1815. Humana Press, New York, NY.
• Hasnain, A., Naqvi, S.A.H., Ayesha, S.I., Khalid, F., Ellahi, M., Iqbal, S., Hassan, M.Z., Abbas, A., Adamski, R., Markowska, D., Baazeem, A., Mustafa, G., Moustafa, M., Hasan, M.E., Abdelhamid, M.M.A., 2022. Plants in vitro propagation with its applications in food, pharmaceuticals and cosmetic industries; current scenario and future approaches. Front Plant Sci. 2022 13:1009395.
• Karaismailoğlu, C., 2022. Practicality of seed morpho-anatomical characters for the identification of species Alyssum (Brassicaceae) in Turkey: a systematic approach. Kahramanmaraş Sütçü İmam Üniversitesi Tarım ve Doğa Dergisi. 25 (1):114-124.
• Leljak-Levanic, D., Bauer, N., Mihaljevic, S., Jelaska, S., 2004. Changes in DNA methylation during somatic embryogenesis in Cucurbita pepo L. Plant Cell Rep. 23:120-7.
• Li, Y., Kong, Y., Zhang, Z., Yin, Y., Liu, B., Lv, G., Wang, X., 2014. Phylogeny and biogeography of Alyssum (Brassicaceae) based on nuclear ribosomal ITS DNA sequences. Journal of Genetics. 93(2): 313–323.
• Lira-Medeiros. C.F., Parisod, C., Fernandes, R.A., Mata, C.S., Cardoso, M.A., Ferreira, P.C.G., 2010. Epigenetic variation in mangrove plants occurring in contrasting natural environment. PLOS One. 5(4): e10326.
• Makowczyńska, J., Andrzejewska-Golec, E., Sliwinska, E., 2008. Nuclear DNA content in different plant materials of Plantago asiatica L. cultured in vitro. Plant Cell Tiss Organ Cult. 94: 65–71.
• Mirzaei, L., Yadollahi, A., Kermani, M.J., Naderpour, M., Zeinanloo, A.A., Farsi, M., Davoodi D., 2021. Evaluation of genetic stability in olive callus-induced and meristem-induced shoots using flow cytometry and amplified fragment length polymorphism techniques. Plant Methods. 17: 31.
• Murashige T., Skoog F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture, Physiol. Plant. 15: 473-497.
• Ohri, D., 1998. Genome size variation and plant systematics. Annals of Botany. 82 (Supplement A): 75–83.
• Orcan, N., 2002. Alyssum penjwinensis Dudley (Brassicaceae): A new record for the flora of Turkey. Bot. Chron. 15: 31–34.
• Orcan, N., 2006. Alyssum mughlaei (Brassicaceae) a new species from Southwest Anatolia. Nord. J. Bot. 23: 703–705.
• Orcan, N., Binzet, R., 2006. A new record for the Flora of Turkey: Alyssum idaeum Boiss. & Heldr. Pak. J. Bot. 38: 931–933.
• Orcan, N., Mısırdalı, H., 2000. Alyssum lenense Adams (Brassicaceae): A new record for the flora of Turkey. Bot. Chron. 13: 409–412.
• Parzymies, M., Pogorzelec, M., Głębocka, K., Sliwinska, E., 2023. Micropropagation protocol and genetic stability of the Salix myrtilloides plants cultivated in vitro. Biology .12: 168.
• Rabinowicz, P.D., Bennetzen, J.L., 2006. The maize genome as a model for efficient sequence analysis of large plant genomes. Current Opinion in Plant Biology. 9(2): 149–156.
• Raji, M.R., Farajpour M., 2021. Genetic fidelity of regenerated plants via shoot regeneration of muskmelon by inter simple sequence repeat and flow cytometry. Journal of the Saudi Society of Agricultural Sciences. 20(2): 88-93.
• Reeves, R.D., Adıgüzel, N., 2008. The nickel hyperaccumulating plants of the serpentines of Turkey and adjacent areas: A review with new Data. Turkish Journal of Biology. 32(3): 143-153.
• Suda, J., Kron, P., Husband, B.C., Trávníček, P., 2007. Flow cytometry and ploidy: applications in plant systematics, ecology and evolutionary biology. In Flow Cytometry with Plant Cells (eds. Doležel, J., Greilhuber, J. & Suda, J.) 103–130 (Wiley-VCH, Weinheim, Germany, 2007).
• Temel, A., Kartal, G., Gozukırmızı, N., 2008. Genetic and epigenetic variations in barley calli cultures. Biotechnol Biotechnol Equip. 22(4): 911-14.
• Tozyılmaz, V., Bülbül, A.S., Ceylan, Y. 2021. Determination of Antimicrobial, Antioxidant and Antibiofilm Activity of Some Alyssum L. Species in Anatolian Flora. Kahramanmaraş Sütçü İmam Üniversitesi Tarım ve Doğa Dergisi. 24 (4): 715-724.
• Van Hoewyk, D., Taskin, M.B., Yaprak, A.E., Turgay, O.C., Ergul, A., 2018. Profiling of proteasome activity in Alyssum species on serpentine soils in Turkey reveals possible insight into nickel tolerance and accumulation. Plant Physiol. Biochem.124: 184–189.
• Yeşilyurt, E.B., Akaydın, G., 2012. Endemic plants and their threat categories of Muğla province (Turkey) Muğla İli Endemik Bitkileri ve Tehlike Kategorileri (Türkiye). Hacettepe J. Biol. & Chem. 40 (2): 195–212.
• Yılmaz, Ö., 2012. Alyssum kaynakiae sp. nov. (sect. Gamosepalum, Brassicaceae) from southwest Anatolia, Turkey. Nordic Journal of Botany. 30:190–194.
• Yücel, G., Cambaz, E., Çördük, N., 2023. Determination of nuclear DNA content and chromosome number of Verbascum scamandri Murb. (Scrophulariaceae). Anadolu Journal of Agricultural Sciences. 38 (2): 397-406.