Pachytene Chromosome Preparation in Populus deltoides Marsh

Haoyang Xin1, Yue Lan1, Jisen Shi1, Yan Ma2, Mengli Xi1

1 Co‐Innovation Center for Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing, 2 College of Horticulture, Jinling Institute of Technology, Nanjing
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20036
Online Posting Date:  December, 2016
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A technique that produces large numbers of good quality pachytene chromosome preparations has been developed for Populus. Anthers at the pachytene stage of meiosis are used as materials. There are two main modifications in our method relative to the traditional squashing method that address the challenges the thick cytoplasm observed during the pachytene phase of meiosis presents. One is the temperature during squashing, i.e., the slide is placed on a 52°C heater for squashing. The other is the removal of the cover slip using 45% acetic acid. © 2016 by John Wiley & Sons, Inc.

Keywords: modified squash method; Populus deltoides Marsh; pachytene chromosome preparation

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • Male flower buds of Populus deltoides Marsh
  • Distilled water
  • Carbol fuchsin solution (see recipe)
  • Carnoy's I fixative (see recipe)
  • 70% ethanol
  • 45% (v/v) acetic acid
  • Bucket (34 × 31 cm)
  • 1.5‐ml microcentrifuge tubes (Axygen, cat. no. 31108051)
  • 2 to 20 μl and 20 to 200 μl pipets (Rainin, cat. no. 17003619 and 17003622)
  • Dissection needles (135 mm)
  • Stereoscope (Leica, MZ 950)
  • Cover slip (22 × 40 mm; Thermo Fisher Scientific, cat. no. 12545 C)
  • Forceps (100 mm in length)
  • Double‐edged razor blade (38 × 21 mm)
  • Petri dish (35 × 12 mm, sterile; Thermo Fisher Scientific, cat. no. 310109009)
  • Microscope slide (25.4 × 76.2 mm; 1.2 mm thick; Sail Brand, cat. no. 7105)
  • Mouse mat (25 × 30 cm; 4 mm thick)
  • Heating block (Leica, cat. no. 042321474 C)
  • Microscope (Olympus, BX41)
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Literature Cited

Literature Cited
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