Live‐Cell Imaging of Meiotic Spindle and Chromosome Dynamics in Maize (Zea mays)

Natalie J. Nannas1, R. Kelly Dawe1

1 Department of Plant Biology and Department of Genetics, University of Georgia, Athens
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20035
Online Posting Date:  December, 2016
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Abstract

Live‐cell imaging is a powerful tool that allows investigators to directly observe the dynamics of cellular processes. Live imaging has proven particularly useful in studying mitotic and meiotic chromosome segregation, where the assembly of spindles and movement of chromosomes can be quantified in ways not possible with fixed cells. This protocol describes how to image live meiosis in the agriculturally important plant, maize. The creation of fluorescently tagged tubulin allows visualization of maize spindles, and nucleic acid dyestain chromosomes. This protocol describes all steps required for live imaging, including how to grow plants, screen for relevant genotypes, harvest meiotic cells, and collect live movies of meiosis. While this protocol was developed for imaging fluorescently tagged tubulin, it can be easily modified to observe the meiotic dynamics of any fluorescently labeled protein of interest. © 2016 by John Wiley & Sons, Inc.

Keywords: meiosis; chromosome; spindle; microscopy; maize

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Live‐Cell Imaging of Maize Meiosis
  • Alternate Protocol 1: Genotyping Plants by PCR
  • Support Protocol 1: Pine Bark Soil and Fertilization
  • Support Protocol 2: Live Imaging Medium
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Live‐Cell Imaging of Maize Meiosis

  Materials
  • Maize seeds containing fluorescent marker (Maize Cell Genomics Database)
  • Fertilized potting/starting soil (e.g., Sungro Horticulture)
  • Deionized, sterile water
  • Glufosinate herbicide: Finale 11.3%, diluted 1:50 with water (Bayer Crop Science)
  • Pine bark soil (see recipe; protocol 3)
  • Live imaging medium (see recipe; protocol 4)
  • SYTO12 (Molecular Probes, ThermoFisher Scientific)
  • 1 mg/ml poly‐L‐lysine
  • Sterile glassware
  • 6.5‐cm × 6.5‐cm plant containers
  • Growth room or growth lights
  • Gloves
  • Sharpie marker
  • Paper towels and Kimwipes
  • Large plant pots, 5 to 8 gallon
  • Surgical scalpel blade, no. 15
  • Forceps (Dumont no. 55, Fine Science Tools)
  • 35‐mm petri dishes
  • 22 × 40–mm glass coverslips
  • Cotton tip applicators
  • Tupperware or sealable box
  • Dissecting microscope (1 to 4× magnification)
  • Pipets
  • Microscope slides
  • Clear nail polish
  • Fluorescent microscope with camera, objectives (40×, 63×), and software
  • Image analysis software (Slidebook, ImageJ, Fiji, etc.)

Alternate Protocol 1: Genotyping Plants by PCR

  Materials
  • Maize leaf tissue
  • Liquid nitrogen
  • CTAB extraction buffer (see recipe)
  • 24:1 chloroform:isoamyl alcohol
  • 10 mg/ml RNase A
  • Isopropanol
  • 70% (v/v) ethanol
  • 1× TE buffer, pH 8.0
  • PCR master mix (such as Promega 2×MasterMix with Taq)
  • Agarose gel
  • Mortar and pestle
  • Metal spatula, chilled
  • 2‐ml microcentrifuge tubes
  • 65°C water bath
  • Rocker or shaker
  • Pipets and tips
  • Paper towels or Kimwipes
  • Microcentrifuge
  • Dry vacuum centrifuge, optional
  • 30°C incubator, optional
  • Spectrophotometer
  • Thermal cycler
  • Gel electrophoresis apparatus

Support Protocol 1: Pine Bark Soil and Fertilization

  Materials
  • Composted pine bark (Fern Acres Farm)
  • 4‐cubic feet bags of vermiculite (GroSouth)
  • Ground dolomite limestone (GroSouth)
  • Superphosphate (GroSouth)
  • Calcium nitrate
  • Gypsum (GroSouth)
  • Potassium nitrate
  • Micromax micronutrients (GroSouth)

Support Protocol 2: Live Imaging Medium

  Materials
  • L‐Glutamine (Sigma‐Aldrich)
  • L‐Isoleucine (Sigma‐Aldrich)
  • L‐Lysine (Sigma‐Aldrich)
  • L‐Methionine (Sigma‐Aldrich)
  • L‐Threonine (Sigma‐Aldrich)
  • L‐Valine (Sigma‐Aldrich)
  • Deionized, sterile water
  • Ca(NO 3) 2·4H 2O (Sigma‐Aldrich)
  • KCl (Sigma‐Aldrich)
  • KNO 3 (Sigma‐Aldrich)
  • MgSO 4·7H 2O (Sigma‐Aldrich)
  • Na 2SO 4 (Sigma‐Aldrich)
  • NaH 2PO 4·H 2O (Sigma‐Aldrich)
  • CuSO 4·5H 2O (Sigma‐Aldrich)
  • H 3BO 3 (Sigma‐Aldrich)
  • KI (Sigma‐Aldrich)
  • MnSO 4 (Sigma‐Aldrich)
  • ZnSO 4·7H 2O (Sigma‐Aldrich)
  • Glycine (Sigma‐Aldrich)
  • Myo‐inositol (Sigma‐Aldrich)
  • Nicotinic acid (Sigma‐Aldrich)
  • Pyridoxine hydrochloride (Sigma‐Aldrich)
  • Thiamine hydrochloride (Sigma‐Aldrich)
  • FeSO 4·7H 2O (Sigma‐Aldrich)
  • Maltose (C f = 0.1 M) (Sigma‐Aldrich)
  • Sucrose (C f = 0.1 M) (Sigma‐Aldrich)
  • 25 mM n‐propyl gallate (Sigma‐Aldrich)
  • Hydrochloric acid (HCl) (Sigma‐Aldrich)
  • Potassium hydroxide (KOH) (Sigma‐Aldrich)
  • Glassware
  • Scale
  • Autoclave
  • 0.22‐μm filters
  • Stirring hot plate
  • pH meter
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Figures

Videos

Literature Cited

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Internet Resources
  http://maize.jcvi.org/cellgenomics/index.php
  Maize Cell Genomics Database: Database of publicly available maize fluorescent lines including C/Y/RFP‐tubulin line.
  https://www.aphis.usda.gov/aphis/resources/permits
  USDA Animal and Plant Health Inspection Service: The Maize Cell Genomics Database will require you to obtain an interstate transportation permit from the USDA before they send the fluorescent line seeds. File an application for the permit at this Web site.
  http://www.maizegdb.org/
  Maize Genetics and Genomics Database (MaizeGDB): Database of maize resources including the genome sequence with annotated and identified genes, information on mutations and alleles, genetic maps, and other available molecular tools.
  http://www.maizegdb.org/controlled_pollination
  An article at Maize GDB on how to perform a controlled pollination with maize.
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