Metaphase Chromosome Preparation from Soybean (Glycine max) Root Tips

Seth D. Findley1, James A. Birchler2, Gary Stacey1

1 University of Missouri, Division of Plant Sciences, Columbia, Missouri, 2 University of Missouri, Division of Biological Sciences, Columbia, Missouri
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20046
Online Posting Date:  March, 2017
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Abstract

This unit presents a highly reliable protocol to produce and screen metaphase chromosome spreads from root tip cell suspensions of soybean (Glycine max), or other legumes. The procedures represent soybean‐optimized versions of protocols developed for maize. The use of pressurized nitrous oxide to reliably generate metaphase‐arrested chromosomes is crucial to overcoming one of the challenges of working with tiny and numerous soybean chromosomes. © 2017 by John Wiley & Sons, Inc.

Keywords: chromosome structure; fluorescence in situ hybridization (FISH); genomic in situ hybridization (GISH); glycine max; karyotype; legume; metaphase chromosomes; metaphase arrest; nitrous oxide; ploidy

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Soybean Root Tip Chromosome Preparation
  • Alternate Protocol 1: Chromosome Preparation from Other Legume Species
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Soybean Root Tip Chromosome Preparation

  Materials
  • Digestive enzyme cocktail (see recipe)
  • Soybean seeds
  • Diluted household bleach solution (20% v/v, freshly made with sterile distilled or reverse‐osmosis water)
  • Sterile, double‐distilled or reverse‐osmosis water (ddH 2O)
  • GM medium (see recipe)
  • Spray bottle (e.g., Ace Hardware Delta Orbital Sprayer) containing distilled or reverse‐osmosis water
  • Squeeze bottle containing distilled or reverse‐osmosis water
  • 90% glacial acetic acid solution, chilled to 4°C
  • 70% ethanol solution
  • 100% glacial acetic acid
  • Aqueous mounting medium with DAPI (e.g., Vectashield with DAPI, Vector Laboratories)
  • 50‐ml sterile disposable conical polypropylene centrifuge tubes (e.g., Falcon brand)
  • Sterilized forceps for transferring seeds to germination medium
  • Chemical safety hood
  • Tissue culture hood
  • Personal protection equipment (PPE), including gloves, safety glasses and laboratory coat
  • 1.6‐ml microcentrifuge tubes
  • Metal scissors
  • Scalpel with new blades
  • Nitrous oxide pressure treatment chamber (“treatment chamber,” with appropriate fitting for tubing; see https://birchler.biology.missouri.edu/wp‐content/uploads/2015/07/Maize‐Karyotyping‐and‐FISH‐Manual_2015.pdf for details)
  • Nitrous oxide tank, including regulator and HVAC‐type quick connect charging hose
  • Ice bucket
  • Glass or plastic Pasteur pipettes
  • Acetic acid‐resistant marker or pencil
  • Paper towels
  • Metal probe (e.g., Ted Pella, Model 13552)
  • 37°C water bath
  • Rack (e.g., PCR tube rack)
  • Bottomless rack (e.g., P‐1000 box)
  • Microcentrifuge
  • Humidity chamber (e.g., Vollrath 52652 Cutlery Dispenser)
  • Glass slides (e.g., Gold Seal)
  • Plastic wrap or aluminum foil
  • Square glass cover slips
  • Light microscope equipped with DAPI visualization
  • UV cross‐inker (e.g., Ultra‐Violet Products, Model CL‐1000)
  • Slide boxes

Alternate Protocol 1: Chromosome Preparation from Other Legume Species

  Materials
  • Soybean seeds
  • Concentrated sulfuric acid
  • Sterile water
  • GM medium (see recipe)
  • Personal protection equipment (PPE), including gloves, safety glasses and laboratory coat
  • Tissue culture hood
  • 125‐ml Erlenmeyer flasks
  • Sterile forceps
  • 50‐ml disposable conical polypropylene centrifuge tubes
  • 100‐mm petri dishes
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Figures

Videos

Literature Cited

Literature Cited
  Ahmad, Q. N., Britten, E. J., & Byth, D. E. (1983). A quantitative method of karyotypic analysis applied to the soybean. Glycine max. Cytologia, 48, 879–892. doi: http://doi.org/10.1508/cytologia.48.879.
  Ahmad, Q. N., Britten, E. J., & Byth, D. E. (1984). The karyotype of Glycine soja and its relationship to that of the soybean. Glycine max. Cytologia, 49, 645–658. doi: 10.1508/cytologia.49.645.
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  Findley, S. D., Birchler, J. A., & Stacey, G. (2017). Fluorescence in situ hybridization for Glycine max metaphase chromosomes. Current Protocols in Plant Biology, 2, 89–107. doi: 10.1002/cppb.20046.
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