Preparation of Chromosomes from Zea mays

Morgan McCaw1, Nathan Swyers1, Nathaniel Graham1, Jon Cody1, Changzeng Zhao1, James Birchler1

1 University of Missouri, Division of Biological Sciences, Columbia, Missouri
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20032
Online Posting Date:  September, 2016
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Abstract

High‐quality preparations of chromosomes are useful for many purposes. To prepare metaphase chromosome spreads in maize, root tips are harvested and treated with nitrous oxide to stop cell division at metaphase before being fixed in acetic acid. This process allows a high number of condensed chromosome spreads to be obtained at the end of the procedure. To prepare chromosome spreads from various stages of meiosis, anthers are first fixed before being examined for developmental stage. Cells are digested with a mixture of enzymes before the chromosomes are dropped onto glass sides and fixed under UV light. © 2016 by John Wiley & Sons, Inc.

Keywords: fish; maize; Zea mays; karyotyping

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Metaphase Chromosomes
  • Basic Protocol 2: Preparation of Meiotic Chromosomes
  • Support Protocol 1: Determining when to Harvest a Tassel
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Metaphase Chromosomes

  Materials
  • Maize seeds
  • Nitrous oxide gas (local medical gas supply company)
  • 90% glacial acetic acid, store at 4°C
  • 70% ethanol
  • 1× citric buffer (see recipe)
  • Digestive enzyme solution (see recipe)
  • Methanol
  • Ice bucket containing ice
  • Paper towels or Kimwipes
  • 30°C incubator
  • Razor blade or scalpel, tweezers, blunt‐ended stainless steel dissecting probe
  • 0.6‐ or 1.5‐ml tubes
  • Pressure chamber for nitrous oxide treatment (custom manufactured; https://birchler.biology.missouri.edu/wp‐content/uploads/2015/07/Maize‐Karyotyping‐and‐FISH‐Manual_2015.pdf, page 54)
  • Filter paper (Whatman no. 1 or thicker)
  • 37°C water or dry bath
  • Humid chamber
  • Glass microscope slides (1 × 3–in.)
  • Compound microcope
  • UV cross‐linker (120 to 125 mJ/cm2)

Basic Protocol 2: Preparation of Meiotic Chromosomes

  Materials
  • Plant with developing tassel
  • 3:1 fixative: 3 parts 95% ethanol/1 part 100% acetic acid
  • 70% ethanol
  • Acetocarmine stain (see recipe)
  • Alcohol lamp
  • Deionized water or 1× citric buffer (see recipe)
  • Digestive enzyme solution (see recipe)
  • 100% acetic acid or 90% acetic acid
  • 50‐ml tubes
  • Scalpel and forceps
  • Plastic petri dishes
  • Glass slides and 22 × 22–mm glass coverslips
  • Iron dissecting probe
  • Compound light microscope
  • 2‐ml tubes
  • 37°C water bath
  • Lint‐free tissues
  • Blunt‐end, stainless‐steel dissecting probe
  • Humid chamber (plastic chamber with wet paper towels on the bottom and sides with square wooden dowels)
  • UV cross‐linker (total energy of 120 to 125 mJ/cm2)

Support Protocol 1: Determining when to Harvest a Tassel

  Additional Materials (also see protocol 2)
  • Plant with developing tassel
  • 3:1 fixative: 3 parts 95% ethanol: 1 part 100% acetic acid
  • Scalpel and forceps
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Figures

Videos

Literature Cited

  Kato, A., 1999. Air drying method using nitrous oxide for chromosome counting in maize. Biotech. Histochem. 74:160‐166. doi: 10.3109/10520299909047968.
  McCaw, M., Graham, N., Cody, J., Swyers, N., Zhao, C., and Birchler, J. 2016. Fluorescence in situ hybridization to maize (Zea mays) chromosomes. Curr. Protoc. Plant Biol. 1:529‐544. doi: 10.1002/cppb.20033.
  Neuffer, G.M., Coe, E.H., and Wessler, S.R. 1997. Mutants of Maize. Cold Spring Harbor Laboratories Press, Plainview, N.Y.
Internet Resources
  https://birchler.biology.missouri.edu/somatic‐ karyotype‐analysis/
  This site has useful resources related to karyotyping and chromosome preparation, as well as karyotypes for many inbred lines of maize.
  https://www.youtube.com/watch?v=aOX0DrcF‐VE
  The following video demonstrates the procedure and provides helpful hints for success.
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