How to Stain Arabidopsis Meiotic Chromosomes From Pollen Meiocytes

adapted from: Ross, K.J., Fransz, P., Jones, G.H. (1996) A light microscopic atlas of meiosis in Arabidopsis thaliana. Chromosome Research 4:507-516.

This is an excellent article

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I. Solutions and Materials

A. Carnoys Reagent: 60% Ethanol, 30% Chloroform, 10% Acetic Acid

B. 10mM Sodium Citrate pH 4.5

C. DAPI Stain: 1 ug/mL DAPI, 50% Glycerol, 0.1 M Tris pH 9.2, 1 mg/mL Phenylenediamine

E. Cellulase (Sigma C-1184)

F. Pectolyase (Sigma P-3026)

G. Cytohelicase (Sigma C-8274)

II. Proceedure

A. Fix inflorescence in Carnoy's reagent at room temp.: 4 hours with occasional agitation, 8 hours without agitation.

B. Place in -20 for up to a month if you're not going to use immediately

C. 2x 5 minute Water

D. 2x 5 minute Sodium Citrate

E. 2 hours Enzymatic Digestion at 37 degrees in 0.3% Pectolyase, 0.3% cytohelicase, 1% Cellulase.

F. Wash in ice cold Sodium Citrate

G. Dissect anthers from buds

H. Remove excess buffer and add several drops of 60% acetic acid. Macerate anthers with forcepts.

I. Stir anthers on 45 degree hotplate for 1 minute. Don't let the slide dry out, Add more acetic acid if necessary.

J. Flood the slide with ice cold Carnoy's and remove excess liquid.

K. Squash firmly with coverslip. Dry slide with hair dryer.

L. Add DAPI stain and visualize under flourescent microscope.

questions? sbpreuss@ucdavis.edu

DAPI stained telophase chromosomes, They do look better under the scope or at higher resolution.

Section of Plant Biology - University of California at Davis - Davis, CA 95616

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