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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Abstract

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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Materials

Basic Protocol 1: Chemical Mutagenesis of Seed

  Materials
  • 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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Figures

Videos

Literature Cited

 
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