Skip to main content
Log in

Evaluation of ethyl methanesulfonate-induced in vitro mutagenesis, polymorphism and genomic instability in wheat (Triticum aestivum L.)

  • Original Research
  • Published:
Journal of Crop Science and Biotechnology Aims and scope Submit manuscript

Abstract

Wheat (Triticum aestivum L.) is highly rich in nutrients and is an important staple food for humankind. Mutation breeding offers a relatively quick method for crop improvement and it provides variation for selective breeding programs and functional gene studies. In vitro mutagenesis, coupled with in vitro regeneration procedure, can offer a wide variety of plant materials for mutagenesis; enable generation of large mutant populations in a relatively short period. Present experiments were conducted to investigate potential use of conventional chemical mutagenesis technique through ethyl methanesulfonate (EMS) for mature embryo culture in wheat. EMS mutagenesis was experimented with 4 treatment durations (2, 4, 6, and 8 h) and 5 treatment concentrations (0, 0.1, 0.2, 0.3, and 0.4%). Mature embryos were treated to detect optimum doses of mutagenesis and to estimate polymorphism and genomic instability. First of all, 50% reduction in number of regenerated plants as compared to the control (LD50) was adopted as the optimum dose. Treated and untreated mature embryos were transferred to callus induction media. EMS mutagens at different duration and concentration had significant effects on callus formation rate (%), embryogenic callus formation rate (%), responded embryogenic callus rate (%), regeneration efficiency and number of plants parameters. Based on LD50 criterion, the optimum value was achieved at 8 h duration of 0.1% EMS concentration. Secondly, inter-primer binding site (iPBS) markers was applied to investigate insertion polymorphism and genomic instability in the regenerated plants. EMS mutagenic treatments had significant effects on different effects on polymorphism and genomic instability of regenerated plants. Present findings revealed that in vitro mutagenesis might be a useful approach for accelerating breeding strategies to create enough genetic variation in wheat populations. Besides, an integrated approach can be used to carry out mutation-assisted breeding and subsequent selection of desired mutants using molecular markers in wheat.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Arunyanart S, Soontronyatara S (2002) Mutation induction by γ and X-ray irradiation in tissue cultured lotus. Plant Cell Tissue Organ Cult 70(1):119–122

    Article  CAS  Google Scholar 

  • Aydin M, Pour AH, Haliloğlu K, Tosun M (2016) Effect of polyamines on somatic embryogenesis via mature embryo in wheat. Turkish J Biol 40(6):1178–1184

  • Berenschot AS, Zucchi MI, Tulmann-Neto A, Quecini V (2008) Mutagenesis in Petunia × hybrida Vilm. and isolation of a novel morphological mutant. Braz J Plant Physiol 20(2):95–103

    Article  Google Scholar 

  • Bhagwat B, Duncan E (1998) Mutation breeding of banana cv. Highgate (Musa spp., AAA Group) for tolerance to Fusarium oxysporum f. sp. cubense using chemical mutagens. Sci Hortic 73(1):11–22

    Article  CAS  Google Scholar 

  • Bhat TA, Sharma M, Anis M (2007) Comparative analysis of meiotic aberrations induced by diethyl sulphate and sodium azide in broad bean (Vicia faba L.). Asian J Plant Sci 6(7):1051–1055

    Article  CAS  Google Scholar 

  • Cadet J, Douki T, Ravanat JL (2015) Oxidatively generated damage to cellular DNA by UVB and UVA radiation. Photochem Photobiol 91(1):140–155

    Article  CAS  PubMed  Google Scholar 

  • Datta SK (2012) Success story of induced mutagenesis for development of new ornamental varieties. Bioremediat Biodivers Bioavailab 6(1):15–26

    Google Scholar 

  • Delbreil B, Jullien M (1994) Evidence for in vitro induced mutation which improves somatic embryogenesis in Asparagus officinalis L. Plant Cell Rep 13(7):372–376

    Article  CAS  PubMed  Google Scholar 

  • Dobres MS (2008) Barriers to genetically engineered ornamentals: an industry perspective. Floric Ornam Plant Biotechnol 1:14

    Google Scholar 

  • El-Sayed O, Rizkalla A, Sabri S (2007) In vitro mutagenesis for genetic improvement of salinity tolerance in wheat. Res J Agric Biol Sci 4(5):377–383

    Google Scholar 

  • Erturk FA, Agar G, Arslan E, Nardemir G (2015) Analysis of genetic and epigenetic effects of maize seeds in response to heavy metal (Zn) stress. Environ Sci Pollut Res 22(13):10291–10297

    Article  CAS  Google Scholar 

  • Forster BP (2014) A brief chronology and current status of plant mutation breeding. Tagungsband der 64. Jahrestagung der Vereinigung der Pflanzenzüchter und Saatgutkaufleute Österreichs 25.-26. November 2013, Raumberg-Gumpenstein:3

  • Gaj MD (2002) Stimulation of somatic embryo formation by mutagens and darkness in culture of immature zygotic embryos of Arabidopsis thaliana (L.) Heynh. Plant Growth Regul 37(1):93–98

    Article  CAS  Google Scholar 

  • Gbadegesin MA, Wills MA, Beeching JR (2008) Diversity of LTR-retrotransposons and Enhancer/Suppressor Mutator-like transposons in cassava (Manihot esculenta Crantz). Mol Genet Genom 280(4):305

    Article  CAS  Google Scholar 

  • Govindaraju S, Arulselvi PI (2018) Effect of cytokinin combined elicitors (l-phenylalanine, salicylic acid and chitosan) on in vitro propagation, secondary metabolites and molecular characterization of medicinal herb–Coleus aromaticus Benth (L.). J Saudi Soc Agric Sci 17(4):435–444

    Google Scholar 

  • Hayashi K, Yoshida H (2009) Refunctionalization of the ancient rice blast disease resistance gene Pit by the recruitment of a retrotransposon as a promoter. Plant J 57(3):413–425

    Article  CAS  PubMed  Google Scholar 

  • Hofmann N, Raja R, Nelson RL, Korban S (2004) Mutagenesis of embryogenic cultures of soybean and detecting polymorphisms using RAPD markers. Biol Plant 48(2):173–177

    Article  CAS  Google Scholar 

  • Hosseinpour A, Aydin M, Haliloglu K (2019a) Plant regeneration system in recalcitrant rye (Secale cereale L.). Biologia 75:1–12

    Google Scholar 

  • Hosseinpour A, Özkan G, Nalci Ö, Halİloğlu K (2019b) Estimation of genomic instability and DNA methylation due to aluminum (Al) stress in wheat (Triticum aestivum L.) using iPBS and CRED-iPBS analyses. Turk J Bot 43(1):27–37

    Article  Google Scholar 

  • Hosseinpour A, Haliloglu K, Tolga Cinisli K, Ozkan G, Ozturk HI, Pour-Aboughadareh A, Poczai P (2020) Application of zinc oxide nanoparticles and plant growth promoting bacteria reduces genetic impairment under salt stress in tomato (Solanum lycopersicum L. ‘Linda’). Agriculture 10(11):521

    Article  CAS  Google Scholar 

  • Hosseinpour A, Ilhan E, Özkan G, Öztürk Hİ, Haliloglu K, Cinisli KT (2021) Plant growth-promoting bacteria (PGPBs) and copper (II) oxide (CuO) nanoparticle ameliorates DNA damage and DNA Methylation in wheat (Triticum aestivum L.) exposed to NaCl stress. J Plant Biochem Biotechnol 75:1–14

    Google Scholar 

  • Ibrahim R, Mondelaers W, Debergh PC (1998) Effects of X-irradiation on adventitious bud regeneration from in vitro leaf explants of Rosa hybrida. Plant Cell Tissue Organ Cult 54(1):37–44

    Article  Google Scholar 

  • Jain SM (2005) Major mutation-assisted plant breeding programs supported by FAO/IAEA. Plant Cell Tissue Organ Cult 82(1):113–123

    Article  CAS  Google Scholar 

  • Jain SM (2010) Mutagenesis in crop improvement under the climate change. Rom Biotechnol Lett 15(2):88–106

    Google Scholar 

  • Jasdeep P, Avijit T, Varsha S, Harinder V, Gp S, Sanjay S (2019) Cultivar specific response of callus induction and plant regeneration from mature embryos in different elite Indian wheat. Res J Biotechnol 14(2):6

    Google Scholar 

  • Joseph S, Girish T, Nair S, Vasudevan K (1999) Induction and recovery of acyanogenic mutants in cassava. Trop Tuber Crops Food Secur Nutr 124–127

  • Kalendar R, Antonius K, Smýkal P, Schulman AH (2010) iPBS: a universal method for DNA fingerprinting and retrotransposon isolation. Theor Appl Genet 121(8):1419–1430

    Article  CAS  PubMed  Google Scholar 

  • Kannan B, Davila-Olivas NH, Lomba P, Altpeter F (2015) In vitro chemical mutagenesis improves the turf quality of bahiagrass. Plant Cell Tissue Organ Culture PCTOC 120(2):551–561

    Article  CAS  Google Scholar 

  • Kasumi M, Takatsu Y, Manabe T, Hayashi M (2001) The effects of irradiating gladiolus (Gladiolus× grandiflora Hort.) cormels with gamma rays on callus formation, somatic embryogenesis and flower color variations in the regenerated plants. J Jpn Soc Hortic Sci 70(1):126–128

    Article  Google Scholar 

  • Ke C, Guan W, Bu S, Li X, Deng Y, Wei Z, Wu W, Zheng Y (2019) Determination of absorption dose in chemical mutagenesis in plants. PLoS One 14(1):e0210596

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kenganal M, Hanchinal R, Nadaf H (2008) Ethyl methanesulfonate (EMS) induced mutation and selection for salt tolerance in sugarcane in vitro. Indian J Plant Physiol 13:405–410

    CAS  Google Scholar 

  • Khawale RN, Yerramilli V, Singh SK (2007) Molecular marker-assisted selection of in vitro chemical mutagen-induced grapevine mutants. Curr Sci 92(8):1056–1060

    CAS  Google Scholar 

  • Kodym A, Afza R (2003) Physical and chemical mutagenesis plant functional genomics. Springer, New York, pp 189–203

    Book  Google Scholar 

  • Körmöczi P, Tóth B, Nagy-György A, Kocsis K, Óvári J, Szabó B, Véha A, Cseuz L (2019) SNP-based genetic diversity assessment among Hungarian bread wheat (Triticum aestivum L.) genotypes. Cereal Res Commun 48:1–7

    Article  Google Scholar 

  • Kumar K, Gill M, Kaur H, Choudhary O, Gosal S (2010) In vitro mutagenesis and somaclonal variation assisted salt tolerance in “Rough Lemon” (Citrus jambhiri Lush). Eur J Hortic Sci 75(6):233

    Google Scholar 

  • Laneri U, Franconi R, Altavista P (1989) Somatic mutagenesis of Gerbera jamesonii hybr.: irradiation and in vitro culture. In: I International symposium on in vitro culture and horticultural breeding 280, pp 395–402

  • Lee Y-I, Lee I-S, Lim Y-P (2002) Variations in sweet potato regenerates from gamma-ray irradiated embryogenic callus. J Plant Biotechnol 4:163–170

    Google Scholar 

  • Lee IS, Kim DS, Lee SJ, Song HS, Lim YP, Lee YI (2003) Selection and characterizations of radiation-induced salinity-tolerant lines in rice. Breed Sci 53(4):313–318

    Article  CAS  Google Scholar 

  • Ling APK, Chia JY, Hussein S, Harun AR (2008) Physiological responses of Citrus sinensis to gamma irradiation. World Appl Sci J 5(1):12–19

    Google Scholar 

  • Lu G, Zhang X, Zou Y, Zou Q, Xiang X, Cao J (2007) Effect of radiation on regeneration of Chinese narcissus and analysis of genetic variation with AFLP and RAPD markers. Plant Cell Tissue Organ Cult 88(3):319–327

    Article  CAS  Google Scholar 

  • Luan Y-S, Zhang J, Gao X-R, An L-J (2007) Mutation induced by ethylmethanesulphonate (EMS), in vitro screening for salt tolerance and plant regeneration of sweet potato (Ipomoeabatatas L.). Plant Cell Tissue Organ Culture 88(1):77–81

    Article  CAS  Google Scholar 

  • Manzanilla-Ramirez M, Robles-Gonzalez M, Guzman-Gonzalez S, Medina-Urrutia V, Litz R (2001) Radio sensitivity of somatic embryogenic masses of mango cv. Ataulfo (Mangifera indica L.). HortScience 36:535

    Google Scholar 

  • Mehta A, Haber JE (2014) Sources of DNA double-strand breaks and models of recombinational DNA repair. Cold Spring Harb Perspect Biol 6(9):a016428

    Article  PubMed  PubMed Central  Google Scholar 

  • Meriga B, Reddy BK, Rao KR, Reddy LA, Kishor PK (2004) Aluminium-induced production of oxygen radicals, lipid peroxidation and DNA damage in seedlings of rice (Oryza sativa). J Plant Physiol 161(1):63–68

    Article  CAS  PubMed  Google Scholar 

  • Mohd-Yusoff NF, Ruperao P, Tomoyoshi NE, Edwards D, Gresshoff PM, Biswas B, Batley J (2015) Scanning the effects of ethyl methanesulfonate on the whole genome of Lotus japonicus using second-generation sequencing analysis. G3 Genes Genomes Genet 5(4):559–567

    Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15(3):473–497

    Article  CAS  Google Scholar 

  • Ness RW, Morgan AD, Colegrave N, Keightley PD (2012) Estimate of the spontaneous mutation rate in Chlamydomonas reinhardtii. Genetics 192(4):1447–1454

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nonomura T, Ikegami Y, Morikawa Y, Matsuda Y, Toyoda H (2001) Induction of morphologically changed petals from mutagen-treated apical buds of rose and plant regeneration from varied petal-derived calli. Plant Biotechnol 18(3):233–236

    Article  CAS  Google Scholar 

  • Olorunmaiye KS, Joseph G, Animasaun D, Oyedeji S (2019) Mutagenic components and dosage effects of ethyl methanesulphonate on Arachis hypogea (Samnut 24 VR). Ife J Sci 21(2):309–322

    Article  Google Scholar 

  • Omar M, Novak F, Brunner H (1989) In vitro action of ethylmethanesulphonate on banana shoot tips. Sci Hortic 40(4):283–295

    Article  CAS  Google Scholar 

  • Palainivel S (1998) In vitro studies on groundnut Arachis hypogaea L. for crop improvement. Shodhganga@INFLIBNET Bharathidasan University Department of Botany, p 121

  • Picault N, Chaparro C, Piegu B, Stenger W, Formey D, Llauro C, Descombin J, Sabot F, Lasserre E, Meynard D, Guiderdoni E, Panaud O (2009) Identification of an active LTR retrotransposon in rice. Plant J 85:754–765

  • Pozniak CJ, Birk IT, O’Donoughue LS, Ménard C, Hucl PJ, Singh BK (2004) Physiological and molecular characterization of mutation-derived imidazolinone resistance in spring wheat. Crop Sci 44(4):1434–1443

    Article  CAS  Google Scholar 

  • Predieri S (2001) Mutation induction and tissue culture in improving fruits. Plant Cell Tissue Organ Cult 64(2–3):185–210

    Article  CAS  Google Scholar 

  • Predieri S, Di Virgilio N (2007) In vitro mutagenesis and mutant multiplication protocols for micropropagation of woody trees and fruits. Springer, New York, pp 323–333

  • Qin H-M, Wang Y-Q, Hou C-X (2011) Effect of ethyl methane sulfonate (EMS) in in vitro mutation on anther-derived embryos in loquat (Eriobotrya japonica Lindl.). Afr J Agric Res 6(11):2450–2455

    Google Scholar 

  • Saleem M, Mukhtar Z, Cheema A, Atta B (2005) Induced mutation and in vitro techniques as a method to induce salt tolerance in Basmati rice (Oryza sauva L.). Int J Environ Sci Technol 2(2):141–145

    Article  CAS  Google Scholar 

  • Schy WE, Plewa MJ (1989) Molecular dosimetry studies of forward mutation induced at the yg2 locus in maize by ethyl methanesulfonate. Mutat Res Fundam Mol Mech Mutagen 211(2):231–241

    Article  CAS  Google Scholar 

  • Serrat X, Esteban R, Guibourt N, Moysset L, Nogués S, Lalanne E (2014) EMS mutagenesis in mature seed-derived rice calli as a new method for rapidly obtaining TILLING mutant populations. Plant Methods 10(1):5

    Article  PubMed  PubMed Central  Google Scholar 

  • Sharma G, Mirza S, Yang Y-H, Parshad R, Hazrah P, Datta Gupta S, Ralhan R (2009) Prognostic relevance of promoter hypermethylation of multiple genes in breast cancer patients. Anal Cell Pathol 31(6):487–500

    Article  CAS  Google Scholar 

  • Sharma V, Collins LB, Chen T-h, Herr N, Takeda S, Sun W, Swenberg JA, Nakamura J (2016) Oxidative stress at low levels can induce clustered DNA lesions leading to NHEJ mediated mutations. Oncotarget 7(18):25377

    Article  PubMed  PubMed Central  Google Scholar 

  • Shu Q-Y, Forster BP, Nakagawa H, Nakagawa H (2012) Plant mutation breeding and biotechnology. Cabi

  • Spencer-Lopes M, Forster BP & Jankuloski L (2018) Manual on mutation breeding. Food and Agriculture Organization of the United Nations (FAO)

  • Sung ZR (1976) Mutagenesis of cultured plant cells. Genetics 84(1):51–57

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Swanson E, Herrgesell M, Arnoldo M, Sippell D, Wong R (1989) Microspore mutagenesis and selection: canola plants with field tolerance to the imidazolinones. Theor Appl Genet 78(4):525–530

    Article  CAS  PubMed  Google Scholar 

  • Szarejko I, Forster B (2007) Doubled haploidy and induced mutation. Euphytica 158(3):359–370

    Article  Google Scholar 

  • Tadele Z (2016) Mutagenesis and TILLING to dissect gene function in plants. Curr Genom 17(6):499–508

    Article  CAS  Google Scholar 

  • Van K-J, Jang H-J, Jang Y-E, Lee S-H (2008) Regeneration of plants from EMS-treated immature embryo cultures in soybean [Glycine max (L.) Merr.]. J Crop Sci Biotechnol 11(2):119–126

    Google Scholar 

  • Venkatachalam P, Geetha N, Jayabalan N, Saravanababu S (1999) Effect of gamma rays and ethyl methane sulphonate on in vitro regeneration in groundnut (Arachis hypogaea L.). Plant Tissue Cult 9:113–120

    Google Scholar 

  • Viana VE, Pegoraro C, Busanello C, de Oliveira AC (2019) Mutagenesis in rice: the basis for breeding a new super plant. Front Plant Sci 10

  • Wang TL, Uauy C, Robson F, Till B (2012) TILLING in extremis. Plant Biotechnol J 10(7):761–772

    Article  CAS  PubMed  Google Scholar 

  • Xue-Lin L, Zhong-Xu L, Yi-Chun N, Xiao-Ping G, Zhang X-L (2009) Methylation-sensitive amplification polymorphism of epigenetic changes in cotton under salt stress. Acta Agron Sin 35(4):588–596

    Article  Google Scholar 

  • Zeinalzadehtabrizi H, Hosseinpour A, Aydin M, Haliloglu K (2015) A modified genomic DNA extraction method from leaves of sunflower for PCR based analyzes. J Biol Environ Sci 7:222–225

    Google Scholar 

Download references

Acknowledgements

In vitro mutagenesis section in this study presents partial outcomes of Ph.D. thesis of Aras Turkoglu, supported by Ataturk University Scientific Research Projects Department (with the Project No: BAP 2013/143). All of this work was supported by the Scientific and Technological Research Council of Turkey (TUBITAK, Project No: TOVAG 113O940).

Author information

Authors and Affiliations

Authors

Contributions

Conceptualization of research (KH); designing of the experiments (AT, MS, KH); contribution of experimental materials (MS, KH); execution of lab experiments and data collection (AT, KH); analysis of data and interpretation (AT); preparation of manuscript (AT, KH).

Corresponding author

Correspondence to Aras Türkoğlu.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Research involving human participants and/or animals

This article does not contain any studies with human participants or animals performed by any of the authors.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Türkoğlu, A., Tosun, M. & Haliloğlu, K. Evaluation of ethyl methanesulfonate-induced in vitro mutagenesis, polymorphism and genomic instability in wheat (Triticum aestivum L.). J. Crop Sci. Biotechnol. 26, 199–213 (2023). https://doi.org/10.1007/s12892-022-00172-2

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12892-022-00172-2

Keywords

Navigation