Assessment of the genetic stability of indirect shoot organogenesis-derived plantlets of Digitalis trojana Ivanina by flow cytometry and cytological analyses

Nurşen ÇÖRDÜK, Gülru Yücel, Nihan Akıncı, Metin Tuna
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In this study, flow cytometry and cytological analysis was used to evaluate the genetic stability of Digitalis trojana Ivanina plants regenerated via indirect shoot organogenesis. For in vitro propagation, leaf explants were excised from seedlings grown in sterile conditions and cultured MS medium supplemented with 3.0 mg/L BAP, 0.1 mg/L NAA. Shoots and calli were subcultured for a period of 2 weeks for shoot multiplication. For rooting, shoots were separated individually and transferred to MS medium containing 0.1% activated charcoal. Genetic stability of the regenerated plants was assessed by flow cytometry and cytological analyses. Flow cytometric analysis revealed that regenerated plantlets had as 2.82 pg nuclear DNA (2C) and seed-derived plants had on average 2.80 pg/2C. Cytological analysis showed that regenerated plantlets have the same number chromosome with seed-derived plantlets of D. trojana (2n=56). Our results have showed that the plantlets propagated in MS medium with 3 mg/L BAP, 0.1 mg/L NAA didn’t differ genetically from donor plants. Therefore, this system can be effective and suitable for clonal propagation of D. trojana. Our result also confirmed that flow cytometry is fast, easy, accurate and relatively cheap method to determine ploidy of in vitro propagated D. trojana plantlets.

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Albach D.C., H.M. Meudt and B. Oxelman, 2005. Piercing together the ‘‘new’’ Plantaginaceae. Amer J Bot 92:297–315

Araújo, L.G., A.S. Prabhu, M.C. Filippi and L.J. Chaves, 2001. RAPD analysis of blast resistant somaclones from upland rice cultivar IAC 47 for genetic divergence. Plant Cell Tiss Org 67:165–172

Bairu, M.W., A.O. Aremu and J. Van Staden, 2011. Somaclonal variation in plants: Causes and detection methods. Plant Growth Regul 63:147–73

Baltisberger, M. 1991. Cytological investigations of some plants from Turkey. Willdenowia 21: 225–232.

Bennet, M.D., P. Bhandol and I.J. Leitch, 2000 Nuclear DNA amounts in angiosperms and their modern uses—807 new estimates. Ann Bot 86:859–909. doi:10.1006/anbo.2000.1253

Benson, E.E., J.E. Danaher, I.M. Pimbley, C.T. Anderson, J.E. Wake, S. Daley and L.K. Adams, 2000. In vitro micropropagation of Primula scotica:a rare Scottish plant. Biodiversity and Conservation 9:711–726.

Bräuchler, C., H. Meinberg and G. Heubl, 2004. Molecular Phylogeny of the Genera Digitalis L. and Isoplexis (Lindley) Loudon (Veronicaceae) Based on ITS- and trnL-F Sequences. Plant Systematics and Evolution, 248: 111-128

Chinnusamy, V. and J.K. Zhu 2009. Epigenetic regulation of stress responses in plants. Curr Opin Plant Biol 12:133–139

Cooper, C., T. Crowther, B.M. Smith, S. Isaac and H.A. Collin, 2006. Assessment of the response of carrot somaclones to Pythium violae, causal agent of cavity spot. Plant Pathol 55:427–432

Çördük, N. and C. Akı, 2013. Cardenolides in Digitalis trojana Ivanina, an Endemic Plant of Ida Mountain, Turkey. Der Pharma Chemica, 5(6)138-141

Çördük, N. and C. Akı, 2014. In vıtro Çoğaltılan Digitalis trojana Ivanina Türünde Progesteron 5β-reduktaz Geninin İfadesinin Belirlenmesi (In Turkish). 22.Ulusal Biyoloji Kongresi Eskişehir s 1271

Çördük, N. and C. Akı, 2014. The Expression Profiles of Progesterone 5β-Reductase Gene in Digitalis trojana, an Endemic Plant of Kazdagi (Mt. Ida), Turkey 5(11)36-38.

Çördük, N. and C. Akı, 2010. Direct Shoot Organogenesis of Digitalis trojana Ivan. an Endemic Medicinal Herb of Turkey. African Journal of Biotechnology. 9(11), pp. 1587-1591

Dolezel, J., and J. Bartos, 2005. Plant DNA flow cytometry and estimation of nuclear genome size. Ann Bot 95:99-110

Ekim, T., M. Koyuncu, M. Vural, H. Duman, Z. Aytaç and N Adıgüzel 2000. Red Data Book of Turkish Plants (Pteridophyta and Spermatophyta). Foundation for Turkish Nature Conservation and Van Centinential University Press, Ankara.

George, E.F. 1993. Plant propagation by tissue culture, part 1: the technology. Exegetics Ltd, London.

George, E.F. M.A. Hall and G.D. Klerk, 2007. Plant Propagation by Tissue Culture. 3rd. Edition. Springer, 30-43

Haux, J. 1999. Digitoksin is a Potential Anticancer Agent for Several Types of Cancer. Med Hypotheses 53(6):543-548.

Herrera, M.T., M. Cacho, P. Corchete, J. Fernandez-Tarrago, 1990. One Step Shoot Multiplication and Rooting of Digitalis thapsi L. Plant Cell Tiss Org 22:179-182

Johnston, J.S., M.D. Bennett, A.L. Rayburn, D.W. Galbraith, H.J. Price, 1999. Reference standards for determination of DNA content of plant nuclei. Am J Bot 86: 609–613

Karp, A., 1994. Origins, causes and uses of variation in plant tissue cultures. In: Vasil IK, Thorpe TA (eds) Plant cell and tissue culture. Kluwer Academic Publishers, Dordrecht; pp 139-152

Kunitake, H., K. Koreeda, M. Mii, 1995. Morphological and cytological characteristics of protoplast-derived plants of statice (Limonium perezii Hubbard). Scientia Horticulturae 60:305-312

Kurata, N. and Omura T, 1978. Karyotype analysis in rice I. A new method for identifying all chromosome pairs. Jpn J Genet 53:251-255.

Larkin, P. and W. Scowcroft, 1981. Somaclonal variation a novel source of variability from cell cultures for plant improvement. Theoretical and Applied Genetics 60:197-214

Larkin, P.J. 1998. Introduction. In: Jain SM, Brar DS, Ahloowalia BS (eds) Somaclonal variation and induced mutations in crop improvement. Kluwer Academic Publishers, Dordrecht, pp 3–13

Leljak- Levanic D., N. Bauer, S. Mihaljevic and S. Jelaska, 2004. Changes in DNA methylation during somatic embryogenesis in Cucurbita pepo L. Plant Cell Rep 23:120–127

Leva, A.R., R. Petruccelli and L.M.R. Rinaldi, 2012. Somaclonal Variation in Tissue Culture: A Case Study with Olive. Recent Advances in Plant in vitro Culture, Annarita Leva and Laura M. R. Rinaldi (Ed), ISBN: 978-953-51-0787-3, Chapter 7, InTech, DOI:10.5772/50367.

Lira-Medeiros, C.F., C. Parisod, R.A. Fernandes, C.S. Mata, M.A. Cardoso and P.C.G. Ferreira, 2010. Epigenetic variation in mangrove plants occurring in contrasting natural environment. PLOS One 5(4):e10326

Loureiro, J., E. Rodriguez, J. Dolezˇel and C. Santos, 2006. Comparison of four nuclear isolation buffers for plant DNA flow cytometry. Annals of Botany 98: 679–689.

Lungeanu, I. 1973. In IOPB chromosome number reports XLII. Taxon 22: 647-654.

Mallon, R. Rodrıguez-Oubina, J. and M.L. Gonzalez, 2010. In vitro propagation of the endangered plant Centaurea ultreiae: assessment of genetic stability by cytological studies, flow cytometry and RAPD analysis. Plant Cell Tiss Organ Cult 101:31–39

Neelakandan, A.K. and K. Wang, 2012. Recent progress in the understanding of tissue culture-induced genome level changes in plants and potential applications. Plant Cell Rep 31:597–620

Nybom, H., K. Weising and B. Rotter 2014. DNA fingerprinting in botany: past, present, future. Invest Genet 5:1–35

Ochatt, S.J., E.M. Patat-Ochatt and A. Moessner, 2011. Ploidy level determination within the context of in vitro breeding. Plant Cell Tissue Org 104:329–341

Orbović, V., M. Calović, Z. Viloria, B. Nielsen, F. Gmitter, W. Castle and J. Grosser 2008. Analysis of genetic variability in various tissue culture-derived lemon plant populations using RAPD and flow cytometry. Euphytica 161: 329-335

Pasqual, M., L.A.S. Pio, A.

C.L. Oliveira and J.D.R. Soares, 2012. Flow Cytometry Applied in Tissue Culture: Recent Advances in Plant in vitro Culture. Annarita Leva and Laura M R. Rinaldi (Ed), ISBN: 978-953-51-0787-3, Chapter 7, InTech, DOI:10.5772/50986.

Rodriguez-Enriquez, J., H.G. Dickinson and R.T. Grant-Downton, 2011. MicroRNA misregulation: an overlooked factor generating somaclonal variation? Trend Plant Sci 16:242–248

Rout, G.R., S. Samantaray and P. Das (2000) In vitro Manipulation and Propagation of Medicinal Plants. Biotechnology Advances 18:91-120

Sangthong, R., M. Mii, P. Soonthornchainaksaeng and K. Supaibulwatana 2004. Characteristics of the Tetraploid Plant Derived as a Somaclonal Variation in Lilium longiflorum. The I Xth International Symposium on Flower Bulbs Acta Hort pp 673.

Sarasan, V., R. Cripps, M.M. Ramsay, C. Atherton, M. McMichien, G. Prendergast and J.K. Rowntree, 2006. Conservation in vitro of threatened plants-progress in the past decade. In Vitro Cell Dev. Biol Plant. 42: 206-214.

Sharma, S., G. Bryan, M. Winfield and S. Millam, 2007. Stability of potato (Solanum tuberosum L.) plants regenerated via somatic embryos, axillary bud proliferated shoots, microtubers and true potato seeds: a comparative phenotypic, cytogenetic and molecular assessment. Planta 226:1449–1458

Singh, S.R., S. Dalal, R. Singh, A.K., Dhawan and R.K. Kalia 2013. Evaluation of genetic fidelity of in vitro raised plants of Dendrocalamus asper (Shult. & Shult. F.) Backer ex K. Heyne using DNA-based markers. Acta Physiol Plant 35:419–430

Sivanesan, I., 2007. Shoot regeneration and somaclonal variation from leaf callus cultures of Plumbago zeylanica Linn. Asian J Plant Sci 6:83–86

Skirvin, R.M., K.D. McPheeters and M. Norton 1994. Sources and frequency of somaclonal variation. Hort Science 29:1232-1237

Stanišića, M., M. Raspora, S. Ninkovića, S. Miloševića, D. Ćalića, B. Bohanecb, M. Trifunovića, M. Petrića, A. Subotića and S. Jevremovića, 2015. Clonal fidelity of Iris sibirica plants regenerated by somatic embryogenesis and organogenesis in leaf-base culture—RAPD and flow cytometer analyses South African Journal of Botany 96 :42–52

Temel, A., G. Kartal and N. Gözükırmızı (2008) Genetic and epigenetic variations in barley calli cultures. Biotechnol Biotechnol Equip 22(4):911–914.

Thiem, B., M. Kikowska, A. Krawczyk, B. Wieckowska and E. Sliwinska, 2013. Phenolic acid and DNA contents of micropropagated Eryngium planum L. Plant Cell Tissue Org 114:197–206

Thiem, B. and E. Sliwinska, 2003. Flow cytometric analysis of nuclear DNA content in cloudberry (Rubus chamaemorus L.) in vitro cultures. Plant Sci 164:129–134

Tsuchiya, T. and C. Nakamura, 1979. Acetocarmine Squash Method for Observing Sugar Beet Chromosomes. Euphytica 28: 249-256.

Uysal, İ. and M. Öztürk, 1991. Digitalis trojana Ivan. Endemik türünün morfolojisi, anatomisi ve ekolojisi. Anadolu University Journal of Faculty of Arts and Sciences (In Turkısh). 3(1):53-61

Vazquez A.M. 2001. Insight into somaclonal variation. Plant Bios 135:57–62

Verma, S.K., G. Sahin, B. Yucesan, N. Sahbaz, I. Eker, S. Gurel and E. Gurel, 2012. Direct somatic embryogenesis from hypocotyl segments of Digitalis trojana Ivan and subsequent plant regeneration. Ind Crop Prod 40:76–80.

Wang, Q-M.and L. Wang 2012. An evolutionary view of plant tissue culture: somaclonal variation and selection. Plant Cell Rep 31(9):1535–1547

Winnicka, K., K. Bielawski and A Bielawska, 2006. Cardiac glycosides in cancer research and cancer therapy. Acta Pol Pharm 63:109-115

Yakar-Tan, N., 1979. In IOPB chromosome number reports LXV. Taxon 28: 631