Chromosome Preparation for Cytogenetic Analyses in Arabidopsis

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.

Healthy Roots and Leaves: Comparative Genome Structure of Horseradish and Watercress

“…Chromosome spreads from fixed young flower buds containing immature anthers were prepared according to a published protocol (Mandáková and Lysak, 2016a), with minor modifications described in Mandáková et al (2016). Ready-to-use chromosome spreads were checked under a phasecontrast microscope for suitable chromosome figures and the amount of cytoplasm.…”

Human-like telomeres in Zostera marina reveal a mode of transition from the plant to the human telomeric sequences

“…Chromosome spreads from root tips were prepared according to a published protocol 22 . Briefly, selected root tips were rinsed in distilled water (twice for 5 min) and sodium citrate buffer (10 mM sodium citrate, pH 4.8; twice for 5 min), and digested in 0.3% (w/v) cellulase, cytohelicase and pectolyase (all Sigma-Aldrich, St Louis, MO, USA) in citrate buffer at 37 °C for 90 min.…”

Painting of Arabidopsis Chromosomes with Chromosome‐Specific BAC Clones

“…Good-quality chromosome preparations on slides (see Basic Protocols 1 and 2 in Mandáková and Lysak, 2016, for preparing chromosome preparations from root tip meristems and generative organ tissues of Arabidopsis and other Brassicaceae species) 2× saline sodium citrate (SSC, see recipe) RNase solution: 100 μg/ml ribonuclease A (stock of 1 mg/ml in distilled water stored in aliquots at −20°C) 0.1 mg/ml pepsin in 10 mM HCl (see recipe) 70%, 80% and 96% ethanol Vectashield antifade (Vector Laboratories) with 2 μg/ml DAPI (4 ,6-diamidino-2-phenylindole, Sigma), store at 4°C 4% formaldehyde in 2× SSC, freshly prepared BAC clones of Arabidopsis thaliana (Arabidopsis Biological Resource Center, Columbus, OH) 10× NT buffer (see recipe) 3. Tilt slide to allow coverslip to fall off and wash slide as in step 1.…”

“…). The highest resolution of painting signals is achieved by the application of extended meiotic chromosomes at pachytene (see Basic Protocols 1 and 2 in Mandáková and Lysak, ; Fig. ).…”