Chemical Mutagenesis of Seed and Vegetatively Propagated Plants Using EMS

Joanna Jankowicz‐Cieslak1, Bradley J. Till1

1 Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, IAEA Laboratories Seibersdorf, International Atomic Energy Agency, Vienna International Centre, Vienna
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20040
Online Posting Date:  December, 2016
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Chemical mutagenesis provides an inexpensive and straightforward way to generate a high density of novel nucleotide diversity in the genomes of plants and animals. Mutagenesis therefore can be used for functional genomic studies and also for plant breeding. The most commonly used chemical mutagen in plants is ethyl methanesulfonate (EMS). EMS has been shown to induce primarily single base point mutations. Hundreds to thousands of heritable mutations can be induced in a single plant line. A relatively small number of plants, therefore, are needed to produce populations harboring deleterious alleles in most genes. EMS mutagenized plant populations can be screened phenotypically (forward‐genetics), or mutations in genes can be identified in advance of phenotypic characterization (reverse‐genetics). Reverse‐genetics using chemically induced mutations is known as Targeting Induced Local Lesions IN Genomes (TILLING). This unit gives information on EMS treatment of seed and vegetative propagules. © 2016 by John Wiley & Sons, Inc.

Keywords: induced mutation; ethyl methanesulfonate; TILLING; point mutation; tissue culture; forward‐genetics; reverse‐genetics

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Chemical Mutagenesis of Seed
  • Support Protocol 1: Determining Optimal Concentrations of EMS in Seed Mutagenesis Experiments
  • Basic Protocol 2: Chemical Mutagenesis of In Vitro Material
  • Support Protocol 2: Determining Optimal Concentrations of EMS in Tissue Mutagenesis Experiments
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Chemical Mutagenesis of Seed

  • Plant seeds (self‐produced using the chosen genotype, or ordered from a seed stock center)
  • 1 M sodium thiosulfate (Sigma‐Aldrich, cat. no. S8503)
  • EMS (Sigma‐Aldrich, cat. no. M0880)
  • DMSO (Sigma‐Aldrich, cat. no. D4540)
  • Sterile distilled water
  • Plastic beakers
  • Orbital shaker capable of 100 rpm (e.g., IKA, cat. no. KS 260 control)
  • Fume hood
  • Disposable pipettes (10‐ml, or as needed according to calculations in step 6)
  • Graduated cylinders
  • Capped bottles
NOTE: During steps that involve handling EMS, we advise that an assistant be available to provide any necessary equipment (e.g., pipettes) or reagents (e.g., sodium thiosulfate).NOTE: Follow all environmental health and safety guidelines at your institution.

Support Protocol 1: Determining Optimal Concentrations of EMS in Seed Mutagenesis Experiments

  Additional Materials (also see protocol 1)
  • High quality, disease‐free in vitro plantlets
  • Ethanol
  • S‐27 liquid culture medium (see recipe)
  • S‐27 solid culture medium (see recipe)
  • Fume hood
  • Flow bench (for mutagenesis procedure)
  • Flow bench equipped with gas (for post‐mutagenesis propagation)
  • Growth rooms with light and temperature control (light regime 65 µmol/m2/s; e.g., cool white fluorescent tubes, Philips TLP 36/86; temperature regime of 22° ± 2°C)
  • Syringe (size depends on volumes used in step 11)
  • Needle (size depends on volumes used in step 11)
  • Sterile membrane, 25‐mm diameter, 0.2‐µm pore
  • 94‐mm and 145‐mm petri plates
  • 100‐ml and 500‐ml screw‐cap bottles, sterilized
  • 500‐ml and 1000‐ml glass beakers, sterilized
  • 70‐mm metal sieves, 10‐ to 100‐µm pore, sterilized (e.g., 45‐µm pore; Sigma‐Aldrich, cat. no. Z289841)
  • Forceps, sterilized
  • Scalpels, sterilized
  • Scalpel blades
  • Parafilm
  • Orbital shaker
  • 5‐ml and 25‐ml disposable pipettes

Basic Protocol 2: Chemical Mutagenesis of In Vitro Material

  Additional Materials (also see protocol 3)
  • Analytical balance
  • Spreadsheet software e.g., Microsoft Excel, Apache OpenOffice Calc, or LibreOffice Calc
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Literature Cited

  Abe, A., Kosugi, S., Yoshida, K., Natsume, S., Takagi, H., Kanzaki, H., Matsumura, H., Mitsuoka, C., Tamiru, M., Innan, H., Cano, L., Kamoun, S., and Terauchi, R. 2012. Genome sequencing reveals agronomically important loci in rice using MutMap. Nat. Biotechnol. 30:174‐178. doi: 10.1038/nbt.2095.
  Auerbach, C., Robson, J.M., and Carr, J.G. 1947. The chemical production of mutations. Science 105:243‐247. doi: 10.1126/science.105.2723.243.
  Caldwell, D.G., McCallum, N., Shaw, P., Muehlbauer, G.J., Marshall, D.F., and Waugh, R. 2004. A structured mutant population for forward and reverse genetics in barley (Hordeum vulgare L.). Plant J. 40:143‐150. doi: 10.1111/j.1365‐313X.2004.02190.x.
  Chawade, A., Sikora, P., Brautigam, M., Larsson, M., Vivekanand, V., Nakash, M.A., Chen, T., and Olsson, O. 2010. Development and characterization of an oat TILLING‐population and identification of mutations in lignin and beta‐glucan biosynthesis genes. BMC Plant Biol. 10:86. doi: 10.1186/1471‐2229‐10‐86.
  Coen, E.S. and Meyerowitz, E.M. 1991. The war of the whorls: Genetic interactions controlling flower development. Nature 353:31‐37. doi: 10.1038/353031a0.
  Dubcovsky, J., Krasileva, K.V., Vasquez‐Gross, H., Paraiso, F., Howell, T., Wang, X., Clissold, L., Bailey, P.C., Ayling, S., Phillips, A., and Uauy, C. 2016. PAG XXIV: Unravelling hidden variation in the young polyploid wheat genomes. (online), accessed May 2, 2016.
  Gilchrist, E.J., Sidebottom, C.H., Koh, C.S., Macinnes, T., Sharpe, A.G., and Haughn, G.W. 2013. A mutant Brassica napus (canola) population for the identification of new genetic diversity via TILLING and next generation sequencing. PLoS One 8:e84303. doi: 10.1371/journal.pone.0084303.
  Gottwald, S., Bauer, P., Komatsuda, T., Lundqvist, U., and Stein, N. 2009. TILLING in the two‐rowed barley cultivar ‘Barke’ reveals preferred sites of functional diversity in the gene HvHox1. BMC Res. Notes 2:258. doi: 10.1186/1756‐0500‐2‐258.
  Greene, E.A., Codomo, C.A., Taylor, N.E., Henikoff, J.G., Till, B.J., Reynolds, S.H., Enns, L.C., Burtner, C., Johnson, J.E., Odden, A.R., Comai, L., and Henikoff, S. 2003. Spectrum of chemically induced mutations from a large‐scale reverse‐genetic screen in Arabidopsis. Genetics 164:731‐740.
  Hartwell, L.H., Culotti, J., and Reid, B. 1970. Genetic control of the cell‐division cycle in yeast. I. Detection of mutants. Proc. Natl. Acad. Sci. U.S.A. 66:352‐359. doi: 10.1073/pnas.66.2.352.
  Haughn, G.W. and Somerville, C.R. 1987. Selection for herbicide resistance at the whole plant level. In Applications of Biotechnology to Aricultural Chemistry (H.M. LeBaron, R.O. Mumma, R.C. Hoenycutt, and J.H. Duesing, eds.) pp. 98‐108. American Chemical Society, Washington D.C.
  Henry, I.M., Nagalakshmi, U., Lieberman, M.C., Ngo, K.J., Krasileva, K.V., Vasquez‐Gross, H., Akhunova, A., Akhunov, E., Dubcovsky, J., Tai, T.H., and Comai, L. 2014. Efficient genome‐wide detection and cataloging of EMS‐induced mutations using exome capture and next‐generation sequencing. Plant Cell 26:1382‐1397. doi:
  Jankowicz‐Cieslak, J. and Till, B.J. 2015. Forward and reverse genetics in crop breeding. In Advances in Plant Breeding Strategies: Breeding, Biotechnology and Molecular Tools, vol. 1 (J.M. Al‐Khayri, S.M. Jain, and D.V. Johnson, eds.) pp. 215‐240. Springer International Publishing, Switzerland. doi: 10.1007/978‐3‐319‐22521‐0_8
  Jankowicz‐Cieslak, J., Huynh, O.A., Bado, S., Matijevic, M., and Till, B.J. 2011. Reverse‐genetics by TILLING expands through the plant kingdom. Emir. J. Food Agric. 23:290‐300.
  Jankowicz‐Cieslak, J., Huynh, O.A., Brozynska, M., Nakitandwe, J., and Till, B.J. 2012. Induction, rapid fixation and retention of mutations in vegetatively propagated banana. Plant Biotechnol. J. 10:1056‐1066. doi: 10.1111/j.1467‐7652.2012.00733.x.
  Kettleborough, R.N., Busch‐Nentwich, E.M., Harvey, S.A., Dooley, C.M., de Bruijn, E., van Eeden, F., Sealy, I., White, R.J., Herd, C., Nijman, I.J., Fenyes, F., Mehroke, S., Scahill, C., Gibbons, R., Wali, N., Carruthers, S., Hall, A., Yen, J., Cuppen, E., and Stemple, D.L. 2013. A systematic genome‐wide analysis of zebrafish protein‐coding gene function. Nature 496:494‐497. doi: 10.1038/nature11992.
  Knoll, J.E., Ramos, M.L., Zeng, Y.J., Holbrook, C.C., Chow, M., Chen, S.X., Maleki, S., Bhattacharya, A., and Ozias‐Akins, P. 2011. TILLING for allergen reduction and improvement of quality traits in peanut (Arachis hypogaea L.). BMC Plant Biol. 11:81. doi: 10.1186/1471‐2229‐11‐81.
  Kurowska, M., Labocha‐Pawlowska, A., Gnizda, D., Maluszynski, M., and Szarejko, I. 2012. Molecular analysis of point mutations in a barley genome exposed to MNU and gamma rays. Mutat. Res. 738‐739:52‐70. doi: 10.1016/j.mrfmmm.2012.08.008.
  Kurowska, M., Daszkowska‐Golec, A., Gruszka, D., Marzec, M., Szurman, M., Szarejko, I., and Maluszynski, M. 2011. TILLING—a shortcut in functional genomics. J. Appl. Genet. 52:371‐390. doi: 10.1007/s13353‐011‐0061‐1.
  Mba, C., Afza, R., Bado, S., and Jain, S.H. 2010. Induced mutagenesis in plants using physical and chemical agents. In Plant Cell Culture: Essential Methods (M.R. Davey and P. Anthony, eds.) pp. 111‐130. John Wiley & Sons, Ltd.
  McCallum, C.M., Comai, L., Greene, E.A., and Henikoff, S. 2000. Targeted screening for induced mutations. Nat. Biotechnol. 18:455‐457. doi: 10.1038/74542.
  McKey, D., Elias, M., Pujol, B., and Duputie, A. 2010. The evolutionary ecology of clonally propagated domesticated plants. New Phytol. 186:318‐332. doi: 10.1111/j.1469‐8137.2010.03210.x.
  Mei, Y., Wang, Y., Chen, H., Sun, Z.S., and Ju, X.D. 2016. Recent progress in CRISPR/Cas9 technology. J. Genet. Genomics 43:63‐75. doi: 10.1016/j.jgg.2016.01.001.
  Nusslein‐Volhard, C. and Wieschaus, E. 1980. Mutations affecting segment number and polarity in Drosophila. Nature 287:795‐801. doi: 10.1038/287795a0.
  Ossowski, S., Schneeberger, K., Lucas‐Lledo, J.I., Warthmann, N., Clark, R.M., Shaw, R.G., Weigel, D., and Lynch, M. 2010. The rate and molecular spectrum of spontaneous mutations in Arabidopsis thaliana. Science 327:92‐94. doi: 10.1126/science.1180677.
  Rawat, N., Sehgal, S.K., Joshi, A., Rothe, N., Wilson, D.L., McGraw, N., Vadlani, P.V., Li, W., and Gill, B.S. 2012. A diploid wheat TILLING resource for wheat functional genomics. BMC Plant Biol. 12:205. doi: 10.1186/1471‐2229‐12‐205.
  Sabetta, W., Alba, V., Blanco, A., and Montemurro, C. 2011. sunTILL: A TILLING resource for gene function analysis in sunflower. Plant Methods 7:20. doi: 10.1186/1746‐4811‐7‐20.
  Schneeberger, K., Ossowski, S., Lanz, C., Juul, T., Petersen, A.H., Nielsen, K.L., Jorgensen, J.E., Weigel, D., and Andersen, S.U. 2009. SHOREmap: Simultaneous mapping and mutation identification by deep sequencing. Nat. Methods 6:550‐551. doi: 10.1038/nmeth0809‐550.
  Sega, G.A. 1984. A review of the genetic effects of ethyl methanesulfonate. Mutat. Res. 134:113‐142. doi: 10.1016/0165‐1110(84)90007‐1.
  Slade, A.J., Fuerstenberg, S.I., Loeffler, D., Steine, M.N., and Facciotti, D. 2005. A reverse genetic, nontransgenic approach to wheat crop improvement by TILLING. Nat. Biotechnol. 23:75‐81. doi: 10.1038/nbt1043.
  Stadler, L.J. 1929. Chromosome number and the mutation rate in avena and triticum. Proc. Natl. Acad. Sci. U.S.A. 15:876‐881. doi: 10.1073/pnas.15.12.876.
  Suzuki, T., Eiguchi, M., Kumamaru, T., Satoh, H., Matsusaka, H., Moriguchi, K., Nagato, Y., and Kurata, N. 2008. MNU‐induced mutant pools and high performance TILLING enable finding of any gene mutation in rice. Mol. Genet. Genomics 279:213‐223. doi: 10.1007/s00438‐007‐0293‐2.
  Talame, V., Bovina, R., Sanguineti, M.C., Tuberosa, R., Lundqvist, U., and Salvi, S. 2008. TILLMore, a resource for the discovery of chemically induced mutants in barley. Plant Biotechnol. J. 6:477‐485. doi: 10.1111/j.1467‐7652.2008.00341.x.
  Till, B.J., Zerr, T., Comai, L., and Henikoff, S. 2006. A protocol for TILLING and Ecotilling in plants and animals. Nat. Protoc. 1:2465‐2477. doi: 10.1038/nprot.2006.329.
  Till, B.J., Cooper, J., Tai, T.H., Colowit, P., Greene, E.A., Henikoff, S., and Comai, L. 2007. Discovery of chemically induced mutations in rice by TILLING. BMC Plant Biol. 7:19. doi: 10.1186/1471‐2229‐7‐19.
  Till, B.J., Reynolds, S.H., Greene, E.A., Codomo, C.A., Enns, L.C., Johnson, J.E., Burtner, C., Odden, A.R., Young, K., Taylor, N.E., Henikoff, J.G., Comai, L., and Henikoff, S. 2003. Large‐scale discovery of induced point mutations with high‐throughput TILLING. Genome Res. 13:524‐530. doi: 10.1101/gr.977903.
  Uauy, C., Paraiso, F., Colasuonno, P., Tran, R.K., Tsai, H., Berardi, S., Comai, L., and Dubcovsky, J. 2009. A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat. BMC Plant Biol. 9:115. doi: 10.1186/1471‐2229‐9‐115.
  Wang, T.L., Uauy, C., Robson, F., and Till, B. 2012. TILLING in extremis. Plant Biotechnol. J. 10:761‐772. doi: 10.1111/j.1467‐7652.2012.00708.x.
  Xin, Z., Wang, M.L., Barkley, N.A., Burow, G., Franks, C., Pederson, G., and Burke, J. 2008. Applying genotyping (TILLING) and phenotyping analyses to elucidate gene function in a chemically induced sorghum mutant population. BMC Plant Biol. 8:103. doi: 10.1186/1471‐2229‐8‐103.
Internet Resources
  IAEA Mutant Variety Database, accessed May 2, 2016.‐aldrich/docs/Sigma/Product_Information_Sheet/2/m0880pis.pdf
  Ethyl methanesulfonate product information sheet from Sigma‐Aldrich. Accessed May 11, 2016.
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