Chromosome Preparation for Cytogenetic Analyses in Arabidopsis

Terezie Mandáková1, Martin A. Lysak1

1 Central European Institute of Technology (CEITEC), Masaryk University, Brno
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20009
Online Posting Date:  May, 2016
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Abstract

Despite its small nuclear genome and minute chromosomes, the model plant Arabidopsis thaliana has become a well established model in the field of plant cytogenetics. Since 2000, most cytogenetic and epigenetic approaches have been developed for this species and its congeners. In this chapter, we describe two step‐by‐step protocols for preparing chromosome preparations from root tip meristems and generative organ tissues of A. thaliana. Although both protocols have been optimized for A. thaliana, the detailed procedures are equally applicable to other members of the genus Arabidopsis as well as other crucifer species (Brassicaceae). © 2016 by John Wiley & Sons, Inc.

Keywords: arabidopsis; Brassicaceae; mitotic and meiotic chromosomes; pachytene

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Squash Preparation of Chromosomes and Interphase Nuclei from Root Tip Meristems
  • Basic Protocol 2: Surface Spreading Preparation of Chromosomes and Nuclei from Generative Organ Tissues
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Squash Preparation of Chromosomes and Interphase Nuclei from Root Tip Meristems

  Materials
  • Root tips from 2‐ or 3‐day‐old seedlings or potted Arabidopsis plants (Fig.  A)
  • Freshly prepared Carnoy's fixative I (3:1 v/v ethanol: glacial acetic acid)
  • 1× citrate buffer (see recipe for 10×)
  • Pectolytic enzyme mixture (see recipe)
  • 60% (v/v) glacial acetic acid in distilled water
  • Liquid nitrogen or block of dry ice
  • Filter paper
  • Petri dishes
  • Fine‐tipped forceps and long forceps
  • Glass vials or microcentrifuge tubes
  • Black “Assistent” staining blocks with glass cover (Karl Hecht Assistent) or small glass Petri dish
  • Orbital shaker
  • Humid box (e.g., a slide storage box filled with wet paper tissue)
  • Dissecting needles
  • SuperFrost microscope slides (Fisher Scientific)
  • Stereomicroscope
  • 24 × 24 mm glass coverslips
  • Alcohol burner
  • Phase‐contrast microscope
  • Tube rack or a similar holder
  • Dust‐free box for storage of slides
  • Coplin jar

Basic Protocol 2: Surface Spreading Preparation of Chromosomes and Nuclei from Generative Organ Tissues

  Materials
  • Young inflorescences with the majority of closed flower buds containing white anthers (Fig.  B)
  • Freshly prepared Carnoy's fixative I (3:1 v/v ethanol: glacial acetic acid)
  • 70% ethanol
  • 1× citrate buffer (see recipe for 10×)
  • Pectolytic enzyme mixture (see recipe)
  • 60% (v/v) glacial acetic acid
  • Freshly prepared 4% formaldehyde in distilled water (diluted from 37% formaldehyde solution)
  • Fine‐tipped forceps,
  • 15‐ and 50‐ml plastic tubes (e.g., Corning Falcon) or other vials
  • Black “Assistent” staining blocks with glass cover (Karl Hecht Assistent) or small glass Petri dish
  • Stereomicroscope
  • Orbital shaker
  • Humid box (e.g., a slide storage box filled with wet paper tissue)
  • SuperFrost microscope slides (Fisher Scientific)
  • Dissection needles
  • 50°C heating block
  • Hair dryer
  • Phase‐contrast light microscope
  • Coplin jars
  • Dust‐free box for storage of slides
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Figures

Videos

Literature Cited

Literature Cited
  Armstrong, S.J. and Jones, G.H. 2001. Female meiosis in wild‐type Arabidopsis thaliana and in two meiotic mutants. Sex. Plant Reprod. 13:177‐183. doi: 10.1007/s004970000050.
  Mandáková, T. and Lysak, M.A. 2016. Painting of Arabidopsis chromosomes with chromosome‐specific BAC clones. Curr. Protoc. Plant Biol. In press.
  Ross, K.J., Fransz, P., and Jones, G.H. 1996. A light microscopic atlas of meiosis in Arabidopsis thaliana. Chrom. Res. 4:507‐516. doi: 10.1007/BF02261778
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